Gene Literature Dashboard

In the search for new and effective treatments of breast and prostate cancer, a series of hybrid compounds based on tamoxifen, estrogens, and artemisinin were successfully synthesized and analyzed for their in vitro activities against human prostate (PC-3) and breast cancer (MCF-7) cell lines. Most of the hybrid compounds exhibit a strong anticancer activity against both cancer cell lines - for example, EC₅₀ (PC-3) down to 1.07 μM, and EC₅₀ (MCF-7) down to 2.08 μM - thus showing higher activities than their parent compounds 4-hydroxytamoxifen (afimoxifene, 7; EC₅₀ =75.1 (PC-3) and 19.3 μM (MCF-7)), dihydroartemisinin (2; EC₅₀ =263.6 (PC-3) and 49.3 μM (MCF-7)), and artesunic acid (3; EC₅₀ =195.1 (PC-3) and 32.0 μM (MCF-7)). The most potent compounds were the estrogen-artemisinin hybrids 27 and 28 (EC₅₀ =1.18 and 1.07 μM, respectively) against prostate cancer, and hybrid 23 (EC₅₀ =2.08 μM) against breast cancer. These findings demonstrate the high potential of hybridization of artemisinin and estrogens to further improve their anticancer activities and to produce synergistic effects between linked pharmacophores.

Tumour budding in colorectal cancer has become an important prognostic factor. Represented by single cells or small tumour cell clusters at the invasion front of the tumour mass, these tumour buds seem to reflect cells in a 'hybrid' state of epithelial-mesenchymal transition, and evidence indicates that the presence of these entities is associated with lymph node metastasis, local recurrence and distant metastatic disease. The International Tumour Budding Consensus Conference (ITBCC) has highlighted a scoring system for the reporting of tumour budding in colorectal cancer, as well as different clinical scenarios that could affect patient management. Other organs are not spared: tumour budding has been described in numerous gastrointestinal and non-gastrointestinal cancers. Here, we give an update on ITBCC validation studies in the context of colorectal cancer and the clinical implications of tumour budding throughout the upper gastrointestinal and pancreatico-biliary tract.

<h4>Background</h4>Vertebral fractures (VFs) are the most common clinical manifestation of osteoporosis associated with high morbimortality. A personal/familiar history of fractures increases the risk of fractures. The purpose of this study is to identify possible molecular markers associated with osteoporotic VFs in elderly women from community.<h4>Methods</h4>Transcriptomic analysis using Affymetrix HTA2 microarray was performed using whole blood samples of 240 subjects from a population-based survey (Sao Paulo Ageing & Health [SPAH] study). Only elderly women with osteoporosis diagnosis by densitometry were analyzed, and divided in two groups: VF: women with osteoporosis and VFs versus no vertebral fracture (NVF): women with osteoporosis and NVFs. They were matched for age, chronic disease, medication use, and bone mineral density (BMD). The logistic regression model adjusted for age was applied for transcriptome data analysis. SYBR green-based quantitative polymerase chain reaction (qPCR) was used to validate the most significant expression changes obtained in the microarray experiment.<h4>Results</h4>Microarray analysis identified 142 differentially expressed genes (DEGs, p < .01), 57 upregulated and 85 downregulated, compared VF versus NVF groups. The DEG with the greatest expression difference was the Gamma2-Syntrophin (SNTG2) (β = 31.88, p = .005). Validation by qPCR confirmed increased expression in VF group of Syntrophin (SNTG2, fold change = 2.79, p = .009), TRAF3 Interacting Protein2 (TRAF3IP2, fold change = 2.79, p = .020), and Integrin Subunit Alpha 6 (ITGA6, fold change = 2.86, p = .038).<h4>Conclusion</h4>Our data identified and validated the association of SNTG2 (608715), TRAF3IP2 (607043), and ITGA6 (147556) with osteoporotic VF in elderly women, independently of BMD. These results suggest that these transcripts have potential clinical significance and may help to explain the molecular mechanisms and biological functions of vertebral fracture.

The BthA protein from the microorganism <i>Burkholderia thailandensis</i> contains two hemes with axial His/OH₂ and His/Tyr coordinations separated by the closest interheme distance of 14 Å. BthA has a similar structure and belongs to the same family of multiheme cytochrome <i>c</i> peroxidases as MauG, which performs long-range oxidation of the partner protein methylamine dehydrogenase. Magnetic Mössbauer spectroscopy of the diferric state of BthA corroborates previous structural work identifying a high-spin (His/OH₂) peroxidatic heme and a low-spin (His/Tyr) electron transfer heme. Unlike MauG, addition of H₂O₂ fully converts the diferric form of BthA to a stable 2e^- oxidized state, allowing a new assessment of this state. The peroxidatic heme is found to be oxidized to a canonical compound II, S = 1 oxoiron(IV) heme. In contrast, the electronic properties of the oxidized His/Tyr heme are puzzling. The isomer shift of the His/Tyr heme (0.17 mm/s) is close to that of the precursor S = 1/2 Fe³⁺ heme (0.21 mm/s) which suggests oxidation of the Tyr. However, the spin-dipolar hyperfine coupling constants are found here to be the same as those for the ferryl peroxidatic heme, indicating that the His/Tyr heme is also a compound II, S = 1 Fe⁴⁺ heme and ruling out oxidation of the Tyr. DFT calculations indicate that the unusually high isomer shift is not attributable to the rare axial His/Tyr heme coordination. The calculations are only compatible with spectroscopy for an unusually long Fe⁴⁺-O_Tyr distance, which is presumably under the influence of the protein environment of the His/Tyr heme moiety in the H₂O₂ oxidized state of the protein. The results offer new insights into how high valence intermediates can be tuned by the protein environment for performing long-range oxidation.

Risk factors that contribute to inter-individual differences in the age-of-onset of allergic diseases are poorly understood. The aim of this study was to identify genetic risk variants associated with the age at which symptoms of allergic disease first develop, considering information from asthma, hay fever and eczema. Self-reported age-of-onset information was available for 117,130 genotyped individuals of European ancestry from the UK Biobank study. For each individual, we identified the earliest age at which asthma, hay fever and/or eczema was first diagnosed and performed a genome-wide association study (GWAS) of this combined age-of-onset phenotype. We identified 50 variants with a significant independent association (P<3x10-8) with age-of-onset. Forty-five variants had comparable effects on the onset of the three individual diseases and 38 were also associated with allergic disease case-control status in an independent study (n = 222,484). We observed a strong negative genetic correlation between age-of-onset and case-control status of allergic disease (rg = -0.63, P = 4.5x10-61), indicating that cases with early disease onset have a greater burden of allergy risk alleles than those with late disease onset. Subsequently, a multivariate GWAS of age-of-onset and case-control status identified a further 26 associations that were missed by the univariate analyses of age-of-onset or case-control status only. Collectively, of the 76 variants identified, 18 represent novel associations for allergic disease. We identified 81 likely target genes of the 76 associated variants based on information from expression quantitative trait loci (eQTL) and non-synonymous variants, of which we highlight ADAM15, FOSL2, TRIM8, BMPR2, CD200R1, PRKCQ, NOD2, SMAD4, ABCA7 and UBE2L3. Our results support the notion that early and late onset allergic disease have partly distinct genetic architectures, potentially explaining known differences in pathophysiology between individuals.

Skeletal muscle fibers are primarily categorized into oxidative and glycolytic fibers, and the ratios of different myofiber types are important factors in determining livestock meat quality. However, the molecular mechanism for determining muscle fiber types in chickens was hardly understood. In this study, we used RNA sequencing to systematically compare mRNA and microRNA transcriptomes of the oxidative muscle sartorius (SART) and glycolytic muscle pectoralis major (PMM) of Chinese Qingyuan partridge chickens. Among the 44,705 identified mRNAs in the two types of muscles, 3,457 exhibited significantly different expression patterns, including 2,364 up-regulated and 1,093 down-regulated mRNAs in the SART. A total of 698 chicken miRNAs were identified, including 189 novel miRNAs, among which 67 differentially expressed miRNAs containing 42 up-regulated and 25 down-regulated miRNAs in the SART were identified. Furthermore, function enrichment showed that the differentially expressed mRNAs and miRNAs were involved in energy metabolism, muscle contraction, and calcium, peroxisome proliferator-activated receptor (PPAR), insulin and adipocytokine signaling. Using miRNA-mRNA integrated analysis, we identified several candidate miRNA-gene pairs that might affect muscle fiber performance, viz, gga-miR-499-5p/SOX6 and gga-miR-196-5p/CALM1, which were supported by target validation using the dual-luciferase reporter system. This study revealed a mass of candidate genes and miRNAs involved in muscle fiber type determination, which might help understand the molecular mechanism underlying meat quality traits in chickens.

An extensive new multi-proxy database of paleo-temperature time series (Temperature 12k) enables a more robust analysis of global mean surface temperature (GMST) and associated uncertainties than was previously available. We applied five different statistical methods to reconstruct the GMST of the past 12,000 years (Holocene). Each method used different approaches to averaging the globally distributed time series and to characterizing various sources of uncertainty, including proxy temperature, chronology and methodological choices. The results were aggregated to generate a multi-method ensemble of plausible GMST and latitudinal-zone temperature reconstructions with a realistic range of uncertainties. The warmest 200-year-long interval took place around 6500 years ago when GMST was 0.7 °C (0.3, 1.8) warmer than the 19^th Century (median, 5^th, 95^th percentiles). Following the Holocene global thermal maximum, GMST cooled at an average rate -0.08 °C per 1000 years (-0.24, -0.05). The multi-method ensembles and the code used to generate them highlight the utility of the Temperature 12k database, and they are now available for future use by studies aimed at understanding Holocene evolution of the Earth system.

We conducted a cross-trait meta-analysis of genome-wide association study on schizophrenia (SCZ) (n = 65,967), bipolar disorder (BD) (n = 41,653), autism spectrum disorder (ASD) (n = 46,350), attention deficit hyperactivity disorder (ADHD) (n = 55,374), and depression (DEP) (n = 688,809). After the meta-analysis, the number of genomic loci increased from 14 to 19 in ADHD, from 3 to 10 in ASD, from 45 to 57 in DEP, from 8 to 54 in BD, and from 64 to 87 in SCZ. We observed significant enrichment of overlapping genes among different disorders and identified a panel of cross-disorder genes. A total of seven genes were found being commonly associated with four out of five psychiatric conditions, namely GABBR1, GLT8D1, HIST1H1B, HIST1H2BN, HIST1H4L, KCNB1, and DCC. The SORCS3 gene was highlighted due to the fact that it was involved in all the five conditions of study. Analysis of correlations unveiled the existence of two clusters of related psychiatric conditions, SCZ and BD that were separate from the other three traits, and formed another group. Our results may provide a new insight for genetic basis of the five psychiatric disorders.

Myeloid sarcoma (MS) as a solid extra-medullary (EM) manifestation of acute myeloid leukemia (AML), myeloproliferative or myelodysplastic syndromes is a rare presentation of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). The databases of the Departments of Hematology and Oncology of the University Hospitals of Jena and Rostock were screened for patients aged 18 years or older for onset of MS after HSCT for myeloid malignancies between 2002 and 2019. Nineteen patients with MS were identified, the majority of whom had received reduced-intensity conditioning (RIC). The median onset of MS was 425 days after HSCT and the median overall survival since MS was 234 days. Although MS is associated with a poor prognosis, three patients survived more than two years and one more than 11 years after MS onset. These results indicate that RIC protocols may be associated with a higher risk of EM relapse. Since EM relapse occurred in the presence of Graft-versus-host-disease, these observations also demonstrate the limitations of graft-versus-tumor effects after HSCT. In conclusion, occurrence of MS after HSCT is associated with a poor prognosis, as multimodal curative concepts including intensive chemotherapy and another HSCT are often not viable.

Hemochromatosis is a clinical syndrome characterized by iron overload in various organs. We present here a case of type 4 hereditary hemochromatosis due to heterozygous mutation in SLC40A1 gene (p.D157A). SLC40A1 encodes ferroportin, a macromolecule only known as iron exporter from mammalian cells. He first presented symptoms correlated with hypopituitarism. Furthermore, marked hyperferritinemia and high transferrin saturation were revealed in combination with the findings of iron overload in the liver, spleen and pituitary gland by computed tomography and magnetic resonance imaging. Liver biopsy revealed iron deposition in both hepatocytes and Kupffer cells. SLC40A1 mutations are considered to cause wide heterogeneity by various ferroportin mutations. Thus, clinicopathological examinations seem to be very important for diagnosing phenotype of type 4 hemochromatosis in addition to the gene analysis. We diagnosed him as type 4B hereditary hemochromatosis (ferroportin-associated hemochromatosis) by the findings of high transferrin saturation and iron deposition in hepatocytes, and then started iron chelating treatment. We should suspect the possibility of hereditary hemochromatosis even in Japanese with severe iron overload. Although the same mutation in SLC40A1 gene (p.D157A) had been reported to cause "loss of function" phenotype, we considered that the mutation of our case caused "gain of function" phenotype.

Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.

Amiodarone, prescribed for cardiac conditions, including dysrhythmia, is associated with bradycardia. However, few reports have demonstrated that bradycardia is observed when amiodarone is used in combination with sofosbuvir, a direct-acting antiviral drug for chronic hepatitis C. We herein report the case of a male patient in his 70s with chronic atrial fibrillation and prostatic hyperplasia and was also taking amiodarone, who experienced severe bradycardia and syncope a day after taking sofosbuvir and ribavirin for chronic hepatitis C. After discontinuing amiodarone, ribavirin, and sofosbuvir, bradycardia did not recur. Therefore, we must consider drug interactions between amiodarone and sofosbuvir in patients with severe bradycardia.

<h4>Objective</h4>To catalog and characterize the proteome of normal human intervertebral disc (IVD).<h4>Methods</h4>Nine magnetic resonance imaging (MRI) normal IVDs were harvested from 9 different brain dead yet alive voluntary organ donors and were subjected to electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) acquisition.<h4>Results</h4>A total of 1,116 proteins were identified. Functional enrichment analysis tool DAVID ver. 6.8 categorized: extracellular proteins (38%), intracellular (31%), protein-containing complex (13%), organelle (9%), membrane proteins (6%), supramolecular complex (2%), and 1% in the cell junction. Molecular function revealed: binding activity (42%), catalytic activity (31%), regulatory activity (14%), and structural activity (7%). Molecular transducer, transporter, and transcription regulator activity together contributed to 6%. A comparison of the proteins obtained from this study to others in the literature showed a wide variation in content with only 3% of bovine, 5% of murine, 54% of human scoliotic discs, and 10.2% of discs adjacent to lumbar burst fractures common to our study of organ donors. Between proteins reported in scoliosis and lumbar fracture patients, only 13.51% were common, further signifying the contrast amongst the various MRI normal IVD samples.<h4>Conclusion</h4>The proteome of "healthy" human IVDs has been defined, and our results show that proteomic data on IVDs obtained from scoliosis, fracture patients, and cadavers lack normal physiological conditions and should not be used as biological controls despite normal MRI findings. This questions the validity of previous studies that have used such discs as controls for analyzing the pathomechanisms of disc degeneration.

While the global prevalence of obesity has risen among both men and women over the past 40 years, obesity has consistently been more prevalent among women relative to men. Neuroimaging studies have highlighted several potential mechanisms underlying an individual's propensity to become obese, including sex/gender differences. Obesity has been associated with structural, functional, and chemical alterations throughout the brain. Whereas changes in somatosensory regions appear to be associated with obesity in men, reward regions appear to have greater involvement in obesity among women than men. Sex/gender differences have also been observed in the neural response to taste among people with obesity. A more thorough understanding of these neural and behavioral differences will allow for more tailored interventions, including diet suggestions, for the prevention and treatment of obesity.

Aggregation of the misfolded proteins β-amyloid, tau, huntingtin, and α-synuclein is one of the most important steps in the pathology underlying a wide spectrum of neurodegenerative disorders, including the two most common ones-Alzheimer's and Parkinson's disease. Activity and toxicity of these proteins depends on the stage and form of aggregates. Excessive production of free radicals, including reactive oxygen species which lead to oxidative stress, is proven to be involved in the mechanism of pathology in most of neurodegenerative disorders. Both reactive oxygen species and misfolded proteins play a physiological role in the brain, and only deregulation in redox state and aggregation of the proteins leads to pathology. Here, we review the role of misfolded proteins in the activation of ROS production from various sources in neurons and glia. We discuss if free radicals can influence structural changes of the key toxic intermediates and describe the putative mechanisms by which oxidative stress and oligomers may cause neuronal death.

Lung cancer is associated with a high mortality, with around 1.8 million deaths worldwide in 2018. Non-small-cell lung cancer (NSCLC) accounts for around 85% of cases and, despite improvement in the management of NSCLC, most patients are diagnosed at advanced stage and the five-year survival remains around 15%. This highlights a need to identify novel ways to treat the disease to reduce the burden of NSCLC. Long non-coding RNAs (lncRNAs) are non-coding RNA molecules longer than 200 nucleotides in length which play important roles in gene expression and signaling pathways. Recently, lncRNAs were implicated in cancer, where their expression is dysregulated resulting in aberrant functions. LncRNAs were shown to function as both tumor suppressors and oncogenes in a variety of cancer types. Although there are a few well characterized lncRNAs in NSCLC, many lncRNAs remain un-characterized and their mechanisms of action largely unknown. LncRNAs have success as therapies in neurodegenerative diseases, and having a detailed understanding of their function in NSCLC may guide novel therapeutic approaches and strategies. This review discusses the role of lncRNAs in NSCLC tumorigenesis, highlighting their mechanisms of action and their clinical potential.

The dynamic evolution of materials with medical applications, particularly for drug delivery and wound dressing applications, gives impetus to design new proposed materials, among which, hydrogels represent a promising, powerful tool. In this context, multifunctional hydrogels have been obtained from chemically modified chitosan and acrylic polymers as cross-linkers, followed by subsequent conjugation with arginine. The hydrogels were finely tuned considering the variation of the synthetic monomer and the preparation conditions. The advantage of using both natural and synthetic polymers allowed porous networks with superabsorbent behavior, associated with a non-Fickian swelling mechanism. The in vitro release profiles for ibuprofen and the corresponding kinetics were studied, and the results revealed a swelling-controlled release. The biodegradability studies in the presence of lysozyme, along with the hemostatic evaluation and the induced fibroblast and stem cell proliferation, have shown that the prepared hydrogels exhibit characteristics that make them suitable for local drug delivery and wound dressing.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH₂) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH₂ resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the <i>Candida</i> genus (MICs in the 6.25 - 100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH₂ was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH₂ and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC₅₀ values in the 10 - 100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH₂ since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

The present study aims to evaluate effect of hydroxyapatite (HA, Ca₁₀(PO₄)₆OH₂), a ceramic similar to natural bone, into AZ31B Mg alloy matrix on biomineralization and biocompatibility. The novel friction stir processing additive manufacturing route was employed to fabricate Mg-HA composites. Various HA contents (5, 10, 20 wt%) were incorporated into Mg matrix. Microstructural observation and chemical composition analysis revealed that refined Mg grains and dispersion of HA particles at micro/nanoscales were achieved in Mg-HA composites after the friction stir processing. The biomineralization evaluation were carried out using immersion experiments in simulated body fluid followed by mineral morphology observation and chemical composition analysis. The wettability measurements were conducted to correlate the biomineralization behavior. The results showed improvement in wettability and bone-like Ca/P ratio in apatite deposit on the composites compared to as-received Mg. In addition, the increase of blood compatibility, cell viability and spreading were found in the higher HA content composites, indicating the improved biocompatibility. Therefore, friction stir processed Mg-20 wt%HA composite exhibited the highest wettability and better cell adhesion among other composites due to the effect of increased HA content within Mg matrix.

Geranylgeranyl diphosphate synthase (GGDPS) inhibitors are of potential therapeutic interest as a consequence of their activity against the bone marrow cancer multiple myeloma. A series of bisphosphonates linked to an isoprenoid tail through an amide linkage has been prepared and tested for the ability to inhibit GGDPS in enzyme and cell-based assays. The amides were designed as analogues to triazole-based GGDPS inhibitors. Several of the new compounds show GGDPS inhibitory activity in both enzyme and cell assays, with potency dependent on chain length and olefin stereochemistry.

<h4>Background & aims</h4>Systemic iron status affects multiple health outcomes, however its net effect on life expectancy is not known. We conducted a two-sample Mendelian randomization (MR) study to investigate the association of genetically proxied iron status with life expectancy.<h4>Methods</h4>Using genetic data from 48,972 individuals, we identified three genetic variants as instrumental variables for systemic iron status. We obtained genetic associations of these variants with parental lifespan (n = 1,012,240) and individual survival to the 90th vs. 60th percentile age (11,262 cases and 25,483 controls). We used the inverse-variance weighted method to estimate the effect of a 1-standard deviation (SD) increase in genetically predicted serum iron on each of the life expectancy outcomes.<h4>Results</h4>We found a detrimental effect of genetically proxied higher iron status on life expectancy. A 1-SD increase in genetically predicted serum iron corresponded to 0.70 (95% confidence interval [CI] -1.17, -0.24; P = 3.00 × 10^-3) fewer years of parental lifespan and had odds ratio 0.81 (95% CI 0.70, 0.93; P = 4.44 × 10^-3) for survival to the 90th vs. 60th percentile age. We did not find evidence to suggest that these results were biased by pleiotropic effects of the genetic variants.<h4>Conclusions</h4>Higher systemic iron status may reduce life expectancy. The clinical implications of this finding warrant further investigation, particularly in the context of iron supplementation in individuals with normal iron status.

Protocadherins (Pcdhs) are cell adhesion molecules that belong to the cadherin superfamily, and are subdivided into clustered (cPcdhs) and non-clustered Pcdhs (ncPcdhs) in vertebrates. In this review, we summarize their discovery, expression mechanisms, and roles in neuronal development and cancer, thereby highlighting the context-dependent nature of their actions. We furthermore provide an extensive overview of current structural knowledge, and its implications concerning extracellular interactions between cPcdhs, ncPcdhs, and classical cadherins. Next, we survey the known molecular action mechanisms of Pcdhs, emphasizing the regulatory functions of proteolytic processing and domain shedding. In addition, we outline the importance of Pcdh intracellular domains in the regulation of downstream signaling cascades, and we describe putative Pcdh interactions with intracellular molecules including components of the WAVE complex, the Wnt pathway, and apoptotic cascades. Our overview combines molecular interaction data from different contexts, such as neural development and cancer. This comprehensive approach reveals potential common Pcdh signaling hubs, and points out future directions for research. Functional studies of such key factors within the context of neural development might yield innovative insights into the molecular etiology of Pcdh-related neurodevelopmental disorders.

Genetic diagnosis of patients with neurodevelopmental disorders is imperative and a standard clinical practice. Considering the continuous accumulation of data on disease-causing variants, reanalysis of previously established sequencing data is important. Periodic reanalysis of variants with uncertain significance has become mandatory in clinical laboratories. Therefore, to confirm the utility of the reanalysis of targeted gene panel data in clinical laboratories, we re-evaluated the data of two groups of patients who had undergone targeted gene panel testing for neurodevelopmental disorders (<i>n</i> = 116) and epileptic encephalopathy (<i>n</i> = 384). This reanalysis was based on a reannotation process reflecting updated databases. Six (5.2%) and seven (1.8%) new pathogenic or likely pathogenic variants were identified in these two groups, respectively, attributable to the updated guidelines and <i>de novo</i> reports from unrelated patients. Although relatively low, considerable increase in the diagnostic yield was confirmed. We suggest that reanalysis of genetic variants, mainly using changes in databases and updated interpretations, should be implemented as a routine practice in clinical laboratories.

The butyrophilins (BTNs) represent a unique family of immunoglobulin. They were considered to be involved in milk lactation after their discovery in 1981. With the development of research, an increasing number of research revealed that BTNs play important roles in immune regulation [1992-2019]. Our research aimed to summarize the BTN research status and their relationship with lung cancers and breast cancers by bibliometrics and bioinformatics methods. Our results indicate that the researches on immune-regulatory functions of BTNs gradually developed from 1992 to 2006, whereas they increased quickly after 2007. There are international cooperations among 56 countries, of which the United States is the most active one with the highest number of studies as well as highest citations. By coauthorship and cocitation analysis, we showed that Adrian Hayday, who is active in γδ T-cell field, was an active author in BTN publications with average year of 2015 and led a subfield. By keywords co-occurrence analysis, we found that γδ T cell, which is an important cancer immune regulator, is one important hotspot. Finally, we found that several BTN members' expression levels were significantly correlated with prognosis of lung cancer and breast cancer patients. Thus, these BTNs might play immune regulatory effects and could serve as potential biomarkers for cancer.

<b>Introduction:</b> In the last two decades there have been advances in the diagnosis and management of neonatal cholestasis, which may have changed its epidemiology, diagnostic accuracy, outcomes, and survival. Our goal was to characterize these changes over time in our setting. <b>Methods:</b> Retrospective cohort study in a tertiary center, enrolling patients born between January 1985 and October 2019. The cohort was divided into two periods, before (A; <i>n</i> = 67) and after (B; <i>n</i> = 87) the year 2000; and in two groups, according to patient's outcome (favorable, unfavorable). Overall survival and survival with and without orthotopic liver transplant (OLT) were evaluated in the two periods (A and B) and in different subgroups of underlying entities. <b>Results:</b> We found that the age of cholestasis recognition decreased significantly from period A to period B [median 43 days and 22 days, respectively, (<i>p</i> < 0.001)]; the changes in epidemiology were relevant, with a significant decrease in alpha-1-antitrypsin deficiency (<i>p</i> < 0.001) and an increase in transient cholestasis (<i>p</i> = 0.004). A next-generation sequencing (NGS) panel available since mid-2017 was applied to 13 patients with contributory results in 7, but, so far, only in 2 patients led to conclusive diagnosis of underlying entities. The number of cases of idiopathic cholestasis did not vary significantly. Over time there was no significant change in the outcome (<i>p</i> = 0.116). Overall survival and survival without OLT had no significant improvement during the period of observation (in periods A and B, 86 vs. 88%, and 85 vs. 87%, respectively). However, in period B, with OLT we achieved the goal of 100% of survival rate. <b>Conclusions:</b> Our data suggest that transient cholestasis became a very important subset of neonatal cholestasis, requiring specific guidance. The NGS panels can provide important inputs on disease diagnosis but, if applied without strict criteria and expertise, they can open a Pandora's box due to misinterpretation. Despite all the advances in accurate diagnosis and timely management-including early recognition of cholestasis-the improvement in patient outcomes and survival were still not significant.

The eukaryotic histone acetylation cycle is composed of three classes of proteins, histone acetyltransferases (HATs) that add acetyl groups to lysine amino acids, bromodomain (BRD) containing proteins that are one of the most characterized of several protein domains that recognize acetyl-lysine (Kac) and effect downstream function, and histone deacetylases (HDACs) that catalyze the reverse reaction. Dysfunction of selected proteins of these three classes is associated with human disease such as cancer. Additionally, the HATs, BRDs, and HDACs of fungi and parasitic protozoa present potential drug targets. Despite their importance, the function and mechanisms of HATs, BRDs, and HDACs and how they relate to chromatin remodeling (CR) remain incompletely understood. <i>Tetrahymena thermophila</i> (Tt) provides a highly tractable single-celled free-living protozoan model for studying histone acetylation, featuring a massively acetylated somatic genome, a property that was exploited in the identification of the first nuclear/type A HAT Gcn5 in the 1990s. Since then, <i>Tetrahymena</i> remains an under-explored model for the molecular analysis of HATs, BRDs, and HDACs. Studies of HATs, BRDs, and HDACs in <i>Tetrahymena</i> have the potential to reveal the function of HATs and BRDs relevant to both fundamental eukaryotic biology and to the study of disease mechanisms in parasitic protozoa.

<h4>Background/purpose</h4>Intraluminal springs have recently been shown to lengthen segments of intestine in a process known as distraction enterogenesis. We hypothesized that biocompatible springs could be used to lengthen defunctionalized murine small intestine and would lead to identifiable intestinal adaptations at the molecular level.<h4>Methods</h4>Age and weight matched C57BL/6 mice underwent surgical insertion of nitinol spring-loaded capsules into a Roux limb of jejunum. Segment lengths were measured at initial spring placement and at euthanasia after 14 and 21 days. Histology and gene expression of the Roux limb were evaluated at scarification and compared to untreated control segments.<h4>Results</h4>Intestinal segments loaded with compressed springs lengthened an average of 240%, which was significantly longer than control segments loaded with either empty capsules or uncompressed springs. Muscularis thickening was greater in spring-treated mice compared to controls without springs. Crypt depth and Lgr5+ expression was greater in mice that received compressed spring treatments when compared to control groups.<h4>Conclusions</h4>Insertion of a compressed nitinol spring into a Roux limb results in significant intestinal lengthening, smooth muscle thickening, and Lgr5+ expression in a mouse model. The ability to increase small bowel length in a defunctionalized murine model may be used to understand the mechanism of distraction enterogenesis.

Super-enhancers (SEs) are a large cluster of <i>cis</i>-regulatory DNA elements that contain many binding motifs, which master transcription factors and cofactors bind to with high density. SEs usually regulate the expression of genes that can control the cell identity and fate, and SEs can be used to explain the patterns of the expression of cell-specific genes. Hence, it shows great potential for application in the treatment of diseases like cancer. At present, the clinical treatments for osteosarcoma, Ewing sarcoma, and other bone-related diseases remain challenging. The poor prognosis and difficult treatment of these diseases imposes heavy economic burden on patients and society. In recent years, research on SEs with respect to bone-related diseases has attracted increasing attention. In this paper, we first review the identification and functional mechanisms of SEs. Then, we integrate the findings of the emerging studies on SEs in bone-related diseases. Finally, we summarize recent strategies for targeting SEs for the treatment of bone-related diseases. This review aims to provide comprehensive insights into the roles of SEs in bone-related diseases.

Thalassemia is inherited autosomal recessive disorders characterized by reduced rate of hemoglobin synthesis due to a defect in alpha or beta globin chain synthesis. Maldives has a beta thalassemia prevalence rate of 16-18%. Classical symptoms of beta thalassemia are common on those patients who present late for blood transfusion which is common among the south Asian countries due to resource poor situation. This case is a rare case report of commonly occurring phenomenon which has been reported less among south Asian region. Reporting this case will help health worker to manage cases accordingly. A five and half year prior diagnosed case of beta thalassemia at age of 2 years and lost to follow up presented with cough, Dyspnoea, Irritability, fatigue with classic symptom of beta thalassemia. She was managed with blood transfusion and kept on continuous follow up for transfusion and iron overload management.

Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

<h4>Introduction</h4>Traumatic brain injury (TBI) or head injury is one of the leading causes of morbidity and mortality globally. TBI includes a fractured skull as an indicator of insult which can affect the treatment outcome as well. The development of any fracture depends on a combination of factors defining the intrinsic properties of the bone and the extrinsic factors related to the impact. A decrease in bone mass secondary to deficiency of calcium (Ca) and phosphorus (P) can be a significant factor intrinsic to the skull bone, which can modulate the outcome of the impact by increasing the susceptibility of bones towards fractures. We undertook this research to find out whether or not the Ca and <i>P</i> concentration in skull bone has a role to play as an intrinsic factor, in the development of skull fracture following Road Traffic Accidents (RTAs).<h4>Methodology</h4>In this case-control study conducted for two years, we collected 94 bone samples, i.e. 47 each, from skull bones with head injuries following RTA, with (case) and without (control) fracture of the skull. The elemental analyses for the bony concentration of Ca and <i>P</i> in both the groups were then compared using energy dispersive X-ray (EDX). Unpaired <i>t</i>-test and Fisher's exact test was used for statistical analysis.<h4>Results</h4>The elemental analysis of bones provided evidence that suggests that whilst; Ca is the only mineral that appears to have a significant correlation with the development of fracture skull, the overall Ca: <i>P</i> ratio of less than 1.99 increases the chances of skull fracture by 3.9 times.<h4>Conclusions</h4>Both individual bony Ca concentration and Ca: <i>P</i> ratio can be regarded as important intrinsic factors for the development of skull fracture.

<h4>Background</h4>Nonalcoholic fatty liver disease (NAFLD) is common in both prediabetic patients and healthy overweight individuals, yet it remains understudied. This study investigates the effects of hepatic steatosis on fibrosis and evaluates the major predictors of liver injury in prediabetes and whether this damage is reversible with Mediterranean diet and administration of the nutraceutical silymarin.<h4>Methods</h4>First, a case-control study was conducted in which 212 patients with prediabetes, not known to have NAFLD, and 126 healthy controls underwent clinical evaluation, transient elastography with measurement of liver stiffness (LS) and controlled attenuation parameter (CAP). Subsequently, the 212 prediabetic patients were enrolled into a prospective randomized interventional study: 104 were allocated to Mediterranean diet alone while 108 followed Mediterranean diet plus supplementation with silymarin (a flavonolignan complex isolated from <i>Silybum marianum</i> and <i>Morus alba</i>). The administered silymarin dose was 210 mg twice daily for 6 months. Clinical and instrumental evaluations were repeated at the end of the 6 month-study period. Prediabetics were genotyped for patatin like phospholipase domain containing 3 (PNPLA3).<h4>Results</h4>In the case-control study, 29% of prediabetic patients have significant fibrosis defined as LS ≥ 7.9 kPa vs only 3% of controls (<i>p</i> < 0.001). PNPLA3 genotype CG/GG are significantly associated with significant fibrosis LS ≥ 7.9 relative to CC genotype χ2(1) = 76.466, <i>p</i> < 0.001. Binomial regression analysis shows that increase in BMI, ALT and AST are significantly associated with increased likelihood of significant fibrosis (χ2(7) = 191.9, <i>p</i> < .001) prior to intervention. In the randomized interventional study, prediabetics following Mediterranean diet alone (group 1) experienced a significant regression of fibrosis and decrease in ALT, HbA1c, FBS after 6 months (<i>p</i> < 0.001); similar findings were observed in patients following Mediterranean diet plus silymarin regimen (group 2); group 2 had a significant decrease in HbA1c relative to group 1 (95% CI: 37.8-38.6 vs 39.5-40.3, <i>p</i> < 0.001).<h4>Conclusion</h4>PNPLA3 genotype CG/GG and elevated BMI are the major predictors of significant fibrosis in prediabetic patients prior to intervention in this study. Mediterranean diet either alone or with silymarin treatment for 6 months leads to significant regression of liver damage and improvement of the glycemic profile in prediabetic patients. Yet, as combination treatment of silymarin with Mediterranean diet shows significant reduction of HbA1c when compared to diet alone, this suggests that silymarin may exert an independent anti-glycemic action.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly-diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, especially short and medium chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, and mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading, RBCs from COVID-19 patients may be incapable of responding to environmental variations in hemoglobin oxygen saturation when traveling from the lungs to peripheral capillaries and, as such, may have a compromised capacity to transport and deliver oxygen. <h4>Graphical Abstract</h4> <h4>Key Points</h4> COVID-19 promotes oxidation and fragmentation of membrane proteins, including the N-term of band 3 RBCs from COVID-19 patients are characterized by increases in glycolysis and altered lipidomes COVID-19 impacts two critical mechanisms that finely tune red cell membranes and hemoglobin oxygen affinity

SARS-CoV-2 , the etiological agent causing COVID-19, has infected more than 8.7 million people with over 461000 deaths worldwide since its emergence in December 2019. Factors for severe disease, such as diabetes, hypertension and obesity have been identified however, the precise pathogenesis is poorly understood. In order to understand its pathophysiology and to develop effective therapeutic strategies, it is essential to define the prevailing immune cellular subsets. We performed circulating immune cells scRNAseq from five critically ill COVID-19 patients. Immature myeloid populations, such as promyelocytes-myelocytes, metamyelocytes, band neutrophils, monocytoid precursors and activated monocytes predominated. Trajectory with pseudotime analysis supported the finding of immature cell states. Gene ontology showed myeloid cell activation in immune response, DNA and RNA processing, defense response to virus and response to type 1 interferon. Lymphoid lineage was scarce. Our results uncover a transcriptomic profiles related to immature myeloid lineages and suggest the potential induction of trained immunity.

Every year nearly 6 percent of children worldwide are born with a serious congenital malformation, resulting in death or lifelong disability. In the United States, birth defects remain one of the leading causes of infant mortality. Among the common structural congenital defects are conditions known as neural tube defects (NTDs). These are a class of malformation of the brain and spinal cord where the neural tube fails to close during the neurulation. Although NTDs remain among the most pervasive and debilitating of all human developmental anomalies, there is insufficient understanding of their etiology. Previous studies have proposed that complex birth defects like NTDs are likely omnigenic, involving interconnected gene regulatory networks with associated signals throughout the genome. Advances in technologies have allowed researchers to more critically investigate regulatory gene networks in ever increasing detail, informing our understanding of the genetic basis of NTDs. Employing a systematic analysis of these complex birth defects using massively parallel DNA sequencing with stringent bioinformatic algorithms, it is possible to approach a greater level of understanding of the genomic architecture underlying NTDs. Herein, we present a brief overview of different approaches undertaken in our laboratory to dissect out the genetics of susceptibility to NTDs. This involves the use of mouse models to identify candidate genes, as well as large scale whole genome/whole exome (WGS/WES) studies to interrogate the genomic landscape of NTDs. The goal of this research is to elucidate the gene-environment interactions contributing to NTDs, thus encouraging global research efforts in their prevention.

Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base-pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.

Background: Oral cancer is a frequently encountered neoplasm of the head and neck region, being the eighth most common type of human malignancy worldwide. Despite improvement in its control, morbidity and mortality, rates have improved little in the past decades. The present investigations about gene interaction and pathways still could not clear the appearance and development of oral squamous cell carcinoma (OSCC), completely. The aim of this study is to investigate the key genes and microRNAs interaction in OSCC. Materials and Methods: The microarray datasets GSE13601 and GSE98463, including mRNA and miRNA profiles, were extracted from the GEO database and were analyzed using GEO2R. Functional and pathway enrichment analyses were performed by using the DAVID database. The protein-protein interaction (PPI) network was constructed and analyzed using STRING database and Cytoscape software, respectively. Finally, miRDB was applied to predict the targets of the differentially expressed miRNAs (DEMs). Results: Totally, 97 differentially expressed genes (DEGs) were found in OSCC, including 66 up-regulated and 31 down-regulated genes. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that up-regulated genes were significantly enriched in movement of cell or subcellular component, cell adhesion, biological adhesion, cellular localization, apoptotic signaling pathway, while the downregulated genes were enriched in muscle system process and oxidation-reduction process. From the PPI network, the top 10 nodes with the highest degree were detected as hub genes. In addition, 18 DEMs were screened, which included 7 up-regulated and 11 down-regulated miRNAs. STAT1 was potentially targeted by three miRNAs, including has-miR- 6825-5P, has-miR-4495, and has-miR-5580-3P. Conclusion: The roles of DEMs such as hsa-mir-5580-3p in OSCC through interactions with DEGs CD44, ACLY, ACTR3, STAT1, LAMC2 and YWHAZ may offer a suitable candidate biomarker pattern for diagnosis, prognosis and treatment processes in OSCC.

<h4>Background</h4>The objective of this study was to measure skin characteristics in premature (PT), late preterm (LPT), and full-term (FT) neonates compared with adults at two times (T1, T2).<h4>Methods</h4>Skin samples of 61 neonates and 34 adults were analyzed for protein biomarkers, natural moisturizing factor (NMF), and biophysical parameters. Infant groups were: <34 weeks (PT), 34-<37 weeks (LPT), and ≥37 weeks (FT).<h4>Results</h4>Forty proteins were differentially expressed in FT infant skin, 38 in LPT infant skin, and 12 in PT infant skin compared with adult skin at T1. At T2, 40 proteins were differentially expressed in FT infants, 38 in LPT infants, and 54 in PT infants compared with adults. All proteins were increased at both times, except TMG3, S100A7, and PEBP1, and decreased in PTs at T1. The proteins are involved in filaggrin processing, protease inhibition/enzyme regulation, and antimicrobial function. Eight proteins were decreased in PT skin compared with FT skin at T1. LPT and FT proteins were generally comparable at both times. Total NMF was lower in infants than adults at T1, but higher in infants at T2.<h4>Conclusions</h4>Neonates respond to the physiological transitions at birth by upregulating processes that drive the production of lower pH of the skin and water-binding NMF components, prevent protease activity leading to desquamation, and increase the barrier antimicrobial properties.<h4>Impact</h4>Neonates respond to the transitions at birth by upregulating processes that drive the production of lower pH of the skin and NMF, prevent protease activity leading to desquamation, and increase the antimicrobial properties of the barrier. The neonatal epidermal barrier exhibits a markedly different array of protein biomarkers both shortly after birth and 2-3 months later, which are differentially expressed versus adults. The major biomarker-functional classes included filaggrin processing, protease inhibitor/enzyme regulators, antimicrobials, keratins, lipids, and cathepsins. The findings will guide improvement of infant skin care practices, particularly for the most premature infants with the ultimate goals mitigating nosocomial infection.

<h4>Background</h4>Genetic associations link hematopoietic traits and disease end-points, but most causal variants and genes underlying these relationships are unknown. Here, we used genetic colocalization to nominate loci and genes related to shared genetic signal for hematopoietic, cardiovascular, autoimmune, neuropsychiatric, and cancer phenotypes.<h4>Methods</h4>Our aim was to identify colocalization sites for human traits among established genome-wide significant loci. Using genome-wide association study (GWAS) summary statistics, we determined loci where multiple traits colocalized at a false discovery rate < 5%. We then identified quantitative trait loci among colocalization sites to highlight related genes. In addition, we used Mendelian randomization analysis to further investigate certain trait relationships genome-wide.<h4>Results</h4>Our findings recapitulated developmental hematopoietic lineage relationships, identified loci that linked traits with causal genetic relationships, and revealed novel trait associations. Out of 2706 loci with genome-wide significant signal for at least 1 blood trait, we identified 1779 unique sites (66%) with shared genetic signal for 2+ hematologic traits. We could assign some sites to specific developmental cell types during hematopoiesis based on affected traits, including those likely to impact hematopoietic progenitor cells and/or megakaryocyte-erythroid progenitor cells. Through an expanded analysis of 70 human traits, we defined 2+ colocalizing traits at 2123 loci from an analysis of 9852 sites (22%) containing genome-wide significant signal for at least 1 GWAS trait. In addition to variants and genes underlying shared genetic signal between blood traits and disease phenotypes that had been previously related through Mendelian randomization studies, we defined loci and related genes underlying shared signal between eosinophil percentage and eczema. We also identified colocalizing signals in a number of clinically relevant coding mutations, including sites linking PTPN22 with Crohn's disease, NIPA with coronary artery disease and platelet trait variation, and the hemochromatosis gene HFE with altered lipid levels. Finally, we anticipate potential off-target effects on blood traits related novel therapeutic targets, including TRAIL.<h4>Conclusions</h4>Our findings provide a road map for gene validation experiments and novel therapeutics related to hematopoietic development, and offer a rationale for pleiotropic interactions between hematopoietic loci and disease end-points.

Asymmetric cell division (ACD) is an evolutionarily conserved mechanism used by prokaryotes and eukaryotes alike to control cell fate and generate cell diversity. A detailed mechanistic understanding of ACD is therefore necessary to understand cell fate decisions in health and disease. ACD can be manifested in the biased segregation of macromolecules, the differential partitioning of cell organelles, or differences in sibling cell size or shape. These events are usually preceded by and influenced by symmetry breaking events and cell polarization. In this Review, we focus predominantly on cell intrinsic mechanisms and their contribution to cell polarization, ACD and binary cell fate decisions. We discuss examples of polarized systems and detail how polarization is established and, whenever possible, how it contributes to ACD. Established and emerging model organisms will be considered alike, illuminating both well-documented and underexplored forms of polarization and ACD.

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyed <i>MBNL1</i> expression in matched tumor/normal pairs across The Cancer Genome Atlas and found that <i>MBNL1</i> was down-regulated in several common cancers. Down-regulation of <i>MBNL1</i> predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared between <i>MBNL1</i>-low cancers and embryonic stem cells including a <i>MAP2K7∆</i>exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversing <i>MAP2K7</i>∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

The National Lung Screening Trial (NLST) demonstrated that screening with low-dose computed tomography (LDCT) is associated with a 20% reduction in lung cancer mortality. One potential limitation of LDCT screening is overdiagnosis of slow growing and indolent cancers. In this study, peritumoral and intratumoral radiomics was used to identify a vulnerable subset of lung patients associated with poor survival outcomes. Incident lung cancer patients from the NLST were split into training and test cohorts and an external cohort of non-screen detected adenocarcinomas was used for further validation. After removing redundant and non-reproducible radiomics features, backward elimination analyses identified a single model which was subjected to Classification and Regression Tree to stratify patients into three risk-groups based on two radiomics features (NGTDM Busyness and Statistical Root Mean Square [RMS]). The final model was validated in the test cohort and the cohort of non-screen detected adenocarcinomas. Using a radio-genomics dataset, Statistical RMS was significantly associated with FOXF2 gene by both correlation and two-group analyses. Our rigorous approach generated a novel radiomics model that identified a vulnerable high-risk group of early stage patients associated with poor outcomes. These patients may require aggressive follow-up and/or adjuvant therapy to mitigate their poor outcomes.

Imaging genetics offers the possibility of detecting associations between genotype and brain structure as well as function, with effect sizes potentially exceeding correlations between genotype and behavior. However, study results are often limited due to small sample sizes and methodological differences, thus reducing the reliability of findings. The IMAGEN cohort with 2000 young adolescents assessed from the age of 14 onwards tries to eliminate some of these limitations by offering a longitudinal approach and sufficient sample size for analyzing gene-environment interactions on brain structure and function. Here, we give a systematic review of IMAGEN publications since the start of the consortium. We then focus on the specific phenotype 'drug use' to illustrate the potential of the IMAGEN approach. We describe findings with respect to frontocortical, limbic and striatal brain volume, functional activation elicited by reward anticipation, behavioral inhibition, and affective faces, and their respective associations with drug intake. In addition to describing its strengths, we also discuss limitations of the IMAGEN study. Because of the longitudinal design and related attrition, analyses are underpowered for (epi-) genome-wide approaches due to the limited sample size. Estimating the generalizability of results requires replications in independent samples. However, such densely phenotyped longitudinal studies are still rare and alternative internal cross-validation methods (e.g., leave-one out, split-half) are also warranted. In conclusion, the IMAGEN cohort is a unique, very well characterized longitudinal sample, which helped to elucidate neurobiological mechanisms involved in complex behavior and offers the possibility to further disentangle genotype × phenotype interactions.

Skin aging is a specific manifestation of the physiological aging process that occurs in virtually all organisms. In this study, we used data independent acquisition mass spectrometry to perform a comparative analysis of protein expression in volar forearm skin samples from of 20 healthy young and elderly Chinese individuals. Our quantitative proteomic analysis identified a total of 95 differentially expressed proteins (DEPs) in aged skin compared to young skin. Enrichment analyses of these DEPs (57 upregulated and 38 downregulated proteins) based on the GO, KEGG, and KOG databases revealed functional clusters associated with immunity and inflammation, oxidative stress, biosynthesis and metabolism, proteases, cell proliferation, cell differentiation, and apoptosis. We also found that GAPDH, which was downregulated in aged skin samples, was the top hub gene in a protein-protein interaction network analysis. Some of the DEPs identified herein had been previously correlated with aging of the skin and other organs, while others may represent novel age-related entities. Our non-invasive proteomics analysis of human epidermal proteins may guide future research on skin aging to help develop treatments for age-related skin conditions and rejuvenation.

Genistein, a soy-derived phytoestrogen, has been shown to exhibit anti-neoplastic activities in various cancers. Nevertheless, its effects on the elimination of tumor-initiating cells of head and neck cancer (HNC-TICs) remain unclear. Here, we investigated the inhibitory effect of genistein on HNC-TICs and potential mechanisms. Our results demonstrated that genistein lowered the proliferation of HNC-TICs by examining the percentage of ALDH1+ or CD44+ cells. Aside from the downregulation of epithelial-mesenchymal transition (EMT) in HNC-TICs, genistein restricted their tumor propagating capacities in a dose-dependent fashion. Moreover, genistein potentiated cell death caused by three commonly used chemotherapeutic agents (doxorubicin, cisplatin, and 5-FU). Our findings proved that genistein induced ROS production through upregulation of miR-34a, leading to apoptosis in HNC-TICs. The genistein-elicited miR-34a reduced self-renewal, migration, invasion capacities and ALDH1 activity, which may be partly owing to the repression of EMT. Furthermore, we showed that RTCB was a novel target that was negatively regulated by miR-34a and involved in the tumor repressive effect of genistein. Besides, the in vivo study validated that genistein retarded tumor growth through the elevation of miR-34a and suppression of RTCB. These results suggested that genistein-induced miR-34a contributed to the ROS-associated apoptosis and diminished stemness properties via repression of RTCB in HNC-TICs.

Primary central nervous system lymphomas (PCNSL) account for approximately 2% to 3% of all primary brain tumors. Until now, neuropathological tumor tissue analysis, most frequently gained by stereotactic biopsy, is still the diagnostic gold standard. Here, we rigorously analyzed two independent patient cohorts comprising the clinical entities PCNSL (<i>n</i> = 47), secondary central nervous system lymphomas (SCNSL; <i>n</i> = 13), multiple sclerosis (MS, <i>n</i> = 23), glioma (<i>n</i> = 10), other tumors (<i>n</i> = 17) and tumor-free controls (<i>n</i> = 21) by proteomic approaches. In total, we identified more than 1220 proteins in the cerebrospinal fluid (CSF) and validated eight candidate biomarkers by a peptide-centric approach in an independent patient cohort (<i>n</i> = 63). Thus, we obtained excellent diagnostic accuracy for the stratification between PCNSL, MS and glioma patients as well as tumor-free controls for three peptides originating from the three proteins VSIG4, GPNMB4 and APOC2. The combination of all three biomarker candidates resulted in diagnostic accuracy with an area under the curve (AUC) of 0.901 (PCNSL vs. MS), AUC of 0.953 (PCNSL vs. glioma) and AUC 0.850 (PCNSL vs. tumor-free control). In summary, the determination of VSIG4, GPNMB4 and APOC2 in CSF as novel biomarkers for supporting the diagnosis of PCNSL is suggested.

<h4>Background</h4><i>Tc1/mariner</i> transposons are widespread DNA transposable elements (TEs) that have made important contributions to the evolution of host genomic complexity in metazoans. However, the evolution and diversity of the <i>Tc1/mariner</i> superfamily remains poorly understood. Following recent developments in genome sequencing and the availability of a wealth of new genomes, <i>Tc1/mariner</i> TEs have been identified in many new taxa across the eukaryotic tree of life. To date, the majority of studies focussing on <i>Tc1</i>/<i>mariner</i> elements have considered only a single host lineage or just a small number of host lineages. Thus, much remains to be learnt about the evolution of <i>Tc1/mariner</i> TEs by performing analyses that consider elements that originate from across host diversity.<h4>Results</h4>We mined the non-redundant database of NCBI using BLASTp searches, with transposase sequences from a diverse set of reference <i>Tc1/mariner</i> elements as queries. A total of 5158 <i>Tc1/mariner</i> elements were retrieved and used to reconstruct evolutionary relationships within the superfamily. The resulting phylogeny is well resolved and includes several new groups of <i>Tc1/mariner</i> elements. In particular, we identify a new family of plant-genome restricted <i>Tc1/mariner</i> elements, which we call <i>PlantMar</i>. We also show that the <i>pogo</i> family is much larger and more diverse than previously appreciated, and we review evidence for a potential revision of its status to become a separate superfamily.<h4>Conclusions</h4>Our study provides an overview of <i>Tc1-mariner</i> phylogeny and summarises the impressive diversity of <i>Tc1-mariner</i> TEs among sequenced eukaryotes. <i>Tc1/mariner</i> TEs are successful in a wide range of eukaryotes, especially unikonts (the taxonomic supergroup containing Amoebozoa, Opisthokonta, Breviatea, and Apusomonadida). In particular, ecdysozoa, and especially arthropods, emerge as important hosts for <i>Tc1/mariner</i> elements (except the <i>PlantMar</i> family). Meanwhile, the <i>pogo</i> family, which is by far the largest <i>Tc1/mariner</i> family, also includes many elements from fungal and chordate genomes. Moreover, there is evidence of the repeated exaptation of <i>pogo</i> elements in vertebrates, including humans, in addition to the well-known example of <i>CENP-B</i>. Collectively, our findings provide a considerable advancement in understanding of <i>Tc1/mariner</i> elements, and more generally they suggest that much work remains to improve understanding of the diversity and evolution of DNA TEs.

A man in his 50s presented with pitting edema of both lower legs and abdominal distension as his chief complaint. His personal medical history and family history were unremarkable, except that he was a heavy drinker consuming 66 g of alcohol per day and a heavy smoker. Blood tests upon admission showed slight hepatic dysfunction, thrombocytopenia, jaundice, hypoalbuminemia, and decreased coagulability. Tumor marker tests showed elevated levels of CA19-9 and PIVKA-II. Contrast-enhanced computed tomography revealed enhancement of multiple masses predominantly in the right lobe of the liver in the early phase, followed by diffuse enhancement of the entire liver in the delayed phase. Hepatic arteriography demonstrated large hemangioma-like lesions corresponding to the masses revealed by computed tomography. That findings seemed to be cotton wool appearance. On magnetic resonance images, there were multiple mass-like lesions that showed homogeneous or heterogeneous low signal intensity on T1-weighted images, and clearly high signal intensity on T2-weighted images. The findings were atypical and no definite diagnosis could be made. Hepatic failure then rapidly worsened, and the patient died on hospital day 20. Autopsy led to the diagnosis of hepatic angiosarcoma.

<h4>Introduction</h4>Antibody-mediated rejection (ABMR) impacts kidney allograft outcome. The diagnosis is made based on findings from invasive kidney transplant biopsy specimens. The aim of this study was to identify a noninvasive urinary protein biomarker for ABMR after kidney transplantation.<h4>Methods</h4>We performed a multicenter case-control study to identify a urinary biomarker for ABMR (training cohort, <i>n</i> = 249) and an independent, prospective multicenter cohort study for validation (<i>n</i> = 391). We used concomitant biopsies to classify the samples according to the Banff classification. After untargeted protein identification and quantification, we used a support vector machine to train the model in the training cohort. The primary endpoint was the diagnostic accuracy of the urinary biomarker for ABMR in the validation cohort.<h4>Results</h4>We identified a set of 10 urinary proteins that accurately discriminated patients with (<i>n</i> = 60) and without (<i>n</i> = 189) ABMR in the training cohort with an area under the curve (AUC) of 0.98 (95% confidence interval [CI], 0.96-1.00). The diagnostic accuracy was maintained in the validation cohort (AUC, 0.88; 95% CI, 0.8-0.93) for discriminating the presence (<i>n</i> = 43) from the absence (<i>n</i> = 348) of ABMR. The negative predictive value of the 10-protein marker set for exclusion of ABMR was 0.99, and the positive predictive value was 0.33. The diagnostic accuracy was independent of the reason for performing the biopsy, time after transplantation, and better than the accuracy of gross proteinuria (AUC, 0.76).<h4>Conclusions</h4>We identified and validated a urinary protein biomarker set that can be used to exclude ABMR.

Efficient variable selection in high dimensional cancer genomic studies is critical for discovering genes associated with specific cancer types and for predicting response to treatment. Censored survival data is prevalent in such studies. In this article we introduce a Bayesian variable selection procedure that uses a mixture prior composed of a point mass at zero and an inverse moment prior in conjunction with the partial likelihood defined by the Cox proportional hazard model. The procedure is implemented in the R package BVSNLP, which supports parallel computing and uses a stochastic search method to explore the model space. Bayesian model averaging is used for prediction. The proposed algorithm provides better performance than other variable selection procedures in simulation studies, and appears to provide more consistent variable selection when applied to actual genomic datasets.

<h4>Introduction</h4>Studies regarding the management of malignant bowel obstruction (MBO) report conflicting findings. This is partly due to different outcome measures being used to evaluate severity of MBO and the response to treatments. Furthermore, current outcome measures focus mainly on measurable physiological parameters which may not correlate strongly with patient-defined quality of life. The development of core outcome sets allows a consistent approach to evaluating clinical conditions taking into consideration patient, healthcare professional and researcher viewpoints. It follows an internationally recognised standard methodology. We present a protocol for the development of a core outcome set for Research and Assessment of MBO (RAMBO).<h4>Methods</h4>RAMBO is a multicentre study, comprising of four phases: a systematic review to examine current scope of outcome measures associated with MBO (phase I). Interviews with patients, companions and healthcare professionals will explore priorities and preferences for care and outcomes (phase II). An expert panel meeting will collate the findings into a set of outcomes (phase III), refined by consensus through a Delphi survey with key stakeholders (phase IV). The final set of outcomes will be ratified at a consensus meeting. Each step will actively include patient partners. Thematic analysis and descriptive statistics will be used to analyse qualitative and quantitative data, respectively.<h4>Ethics and dissemination</h4>Ethical approval was obtained (Wales REC 5, REF: 19/LO/1876). Study participants and relevant stakeholders will be updated with newsletters and a lay summary at the end of the study. Abstracts will be submitted to national and international conferences, result papers will be submitted to peer-reviewed, open access journals.<h4>Trial and prospero registration numbers</h4>Core Outcome Measures in Effectiveness Trials (1402); Systematic Literature Review (CRD42019150648); Rapid Review (CRD42020176393).

<h4>Introduction</h4>Past research has suggested that reading disability is a complex disorder involving genetic and environment contributions, as well as gene-gene and gene-environment interaction, but to date little is known about the underlying mechanisms.<h4>Method</h4>Using the Avon Longitudinal Study of Parents and Children, we assessed the contributions of genetic, demographic, and environmental variables on case-control status using machine learning. We investigated the functional interactions between genes using pathway and network analysis.<h4>Results</h4>Our results support a systems approach to studying the etiology of reading disability with many genes (e.g., RAPGEF2, KIAA0319, DLC1) and biological pathways (e.g., neuron migration, positive regulation of dendrite regulation, nervous system development) interacting with each other. We found that single nucleotide variants within genes often had opposite effects and that enriched biological pathways were mediated by neuron migration. We also identified behavioral (i.e., receptive language, nonverbal intelligence, and vocabulary), demographic (i.e., mother's highest education), and environmental (i.e., birthweight) factors that influenced case-control status when accounting for genetic information.<h4>Discussion</h4>The behavioral and demographic factors were suggested to be protective against reading disability status, while birthweight conveyed risk. We provided supporting evidence that reading disability has a complex biological and environmental etiology and that there may be a shared genetic and neurobiological architecture for reading (dis)ability.

While aging is the greatest risk factor for the development of neurodegenerative disease, the role of aging in these diseases is poorly understood. In the inherited forms of these diseases, the disease-causing mutation is present from birth but symptoms appear decades later. This indicates that these mutations are well tolerated in younger individuals but not in older adults. Based on this observation, we hypothesized that changes taking place during normal aging make the cells in the brain (and elsewhere) susceptible to the disease-causing mutations. If so, then delaying some of these age-related changes may be beneficial in the treatment of neurodegenerative disease. In this review, we examine the effects of five compounds that have been shown to extend longevity (metformin, rapamycin, resveratrol, N-acetyl-l-cysteine, curcumin) in four of the most common neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis). While not all investigations observe a beneficial effect of these compounds, there are multiple studies that show a protective effect of each of these lifespan-extending compounds in animal models of neurodegenerative disease. Combined with genetic studies, this suggests the possibility that targeting the aging process may be an effective strategy to treat neurodegenerative disease.

This meeting report from the XV Banff conference describes the creation of a multiorgan transplant gene panel by the Banff Molecular Diagnostics Working Group (MDWG). This Banff Human Organ Transplant (B-HOT) panel is the culmination of previous work by the MDWG to identify a broadly useful gene panel based on whole transcriptome technology. A data-driven process distilled a gene list from peer-reviewed comprehensive microarray studies that discovered and validated their use in kidney, liver, heart, and lung transplant biopsies. These were supplemented by genes that define relevant cellular pathways and cell types plus 12 reference genes used for normalization. The 770 gene B-HOT panel includes the most pertinent genes related to rejection, tolerance, viral infections, and innate and adaptive immune responses. This commercially available panel uses the NanoString platform, which can quantitate transcripts from formalin-fixed paraffin-embedded samples. The B-HOT panel will facilitate multicenter collaborative clinical research using archival samples and permit the development of an open source large database of standardized analyses, thereby expediting clinical validation studies. The MDWG believes that a pathogenesis and pathway based molecular approach will be valuable for investigators and promote therapeutic decision-making and clinical trials.

Volume-regulated anion channels (VRACs) are critically involved in regulating cell volume, and leucine-rich repeat-containing protein 8A (LRRC8A, SWELL1) is an obligatory subunit of VRACs. Cell swelling occurs early after brain ischemia, but it is unclear whether neuronal LRRC8a contributes to ischemia-induced glutamate release and brain injury. We found that Lrrc8a conditional knockout (Lrrc8a-cKO) mice produced by crossing Nestin^Cre+/- with Lrrc8a^flox+/+ mice died 7-8 weeks of age, indicating an essential role of neuronal LRRC8A for survival. Middle cerebral artery occlusion (MCAO) caused an early increase in LRRC8A protein levels in the hippocampus in wild-type (WT) mice. Whole-cell patch-clamp recording in brain slices revealed that oxygen-glucose deprivation significantly increased the amplitude of VRAC currents in hippocampal CA1 neurons in WT but not in Lrrc8a-cKO mice. Hypotonicity increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in hippocampal CA1 neurons in WT mice, and this was abolished by DCPIB, a VRAC blocker. But in Lrrc8a-cKO mice, hypotonic solution had no effect on the frequency of sEPSCs in these neurons. Furthermore, the brain infarct volume and neurological severity score induced by MCAO were significantly lower in Lrrc8a-cKO mice than in WT mice. In addition, MCAO-induced increases in cleaved caspase-3 and calpain activity, two biochemical markers of neuronal apoptosis and death, in brain tissues were significantly attenuated in Lrrc8a-cKO mice compared with WT mice. These new findings indicate that cerebral ischemia increases neuronal LRRC8A-dependent VRAC activity and that VRACs contribute to increased glutamatergic input to hippocampal neurons and brain injury caused by ischemic stroke.

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (htt^NT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of htt^NT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.

Bicuspid aortic valve (BAV) is the most common congenital heart defect, found in up to 2% of the population and associated with a 30% lifetime risk of complications. BAV is inherited as an autosomal dominant trait with incomplete penetrance and variable expressivity due to a complex genetic architecture that involves many interacting genes. In this review, we highlight the current state of knowledge about BAV genetics, principles and methods for BAV gene discovery, clinical applications of BAV genetics, and important future directions.

A key challenge for neuroscience is to develop generative, causal models of the human nervous system in an individualized, data-driven manner. Previous initiatives have either constructed biologically-plausible models that are not constrained by individual-level human brain activity or used data-driven statistical characterizations of individuals that are not mechanistic. We aim to bridge this gap through the development of a new modeling approach termed Mesoscale Individualized Neurodynamic (MINDy) modeling, wherein we fit nonlinear dynamical systems models directly to human brain imaging data. The MINDy framework is able to produce these data-driven network models for hundreds to thousands of interacting brain regions in just 1-3 ​min per subject. We demonstrate that the models are valid, reliable, and robust. We show that MINDy models are predictive of individualized patterns of resting-state brain dynamical activity. Furthermore, MINDy is better able to uncover the mechanisms underlying individual differences in resting state activity than functional connectivity methods.

Recent advances in single-particle cryogenic-electron microscopy have facilitated an exponential growth in the number of membrane protein structures determined to close to atomic resolution. Nevertheless, despite improvements in microscope hardware, cryo-EM software and sample preparation techniques, challenges remain for structural analysis of small-sized membrane proteins (i.e.<150 kilodalton). Here we discuss recent examples of structures of macromolecules from this category determined by cryo-EM. We analyze the underlying difficulties, the enabling technologies such as the use of antibody fragments to gain size and provide fiducials for particle alignment, and the unresolved issues like dislocation of complexes at the air-water interface. Finally, we briefly highlight the biological relevance of some of these success stories, and our predictions for the future.

Bleeding is the most feared and difficult to predict adverse event of anticoagulation. We sought to investigate whether calibrated automated thrombography (CAT) parameters are associated with minor bleeding (MB) in anticoagulated patients following venous thromboembolism (VTE). Enrolled were 132 patients on rivaroxaban, 145 on vitamin K antagonists (VKA) and 31 controls who stopped anticoagulation. Prior to the next dose of the anticoagulant, we measured CAT parameters, along with rivaroxaban concentration and INR. During a median follow-up of 10 months, we recorded minor and major bleedings. On rivaroxaban, 27 (20.5%) patients with MB had longer time to start thrombin generation, lower peak thrombin generation and lower endogenous thrombin potential compared with subjects without MB (all <i>p</i> < 0.001). All CAT parameters, except for peak thrombin generation (<i>p</i> = 0.049), were similar in VKA patients with (<i>n</i> = 25, 17.2%) vs. without MBs. By logistic regression, time to start thrombin generation (<i>p</i> = 0.007) and unprovoked VTE (<i>p</i> = 0.041) independently predicted MBs on rivaroxaban. Major bleedings were more frequent in patients with MBs (17.3% vs. 1.8%, <i>p</i> < 0.001). Abnormal CAT parameters characterize VTE patients prone to MBs on rivaroxaban, but not on VKA. Time to start thrombin generation measured about 24 h since the last rivaroxaban dose might help predict MBs.

Reactive oxygen species (ROS) are produced predominantly by the mitochondrial electron transport chain and by NADPH oxidases in peroxisomes and in the endoplasmic reticulum. The antioxidative defense counters overproduction of ROS with detoxifying enzymes and molecular scavengers, for instance, superoxide dismutase and glutathione, in order to restore redox homeostasis. Mutations in the redox landscape can induce carcinogenesis, whereas increased ROS production can perpetuate cancer development. Moreover, cancer cells can increase production of antioxidants, leading to resistance against chemo- or radiotherapy. Research has been developing pharmaceuticals to target the redox landscape in cancer. For instance, inhibition of key players in the redox landscape aims to modulate ROS production in order to prevent tumor development or to sensitize cancer cells in radiotherapy. Besides the redox landscape of a single cell, alternative strategies take aim at the multi-cellular level. Extracellular vesicles, such as exosomes, are crucial for the development of the hypoxic tumor microenvironment, and hence are explored as target and as drug delivery systems in cancer therapy. This review summarizes the current pharmaceutical and experimental interventions of the cancer redox landscape.

We previously showed that ergosterol has an inhibitory effect on bladder carcinogenesis. In this study, we aimed to elucidate the molecular mechanism by which ergosterol inhibits bladder carcinogenesis using a rat model of N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer. The messenger ribonucleic acid (mRNA) expression level of the cell cycle-related gene cyclin D1 and inflammation-related gene cyclooxygenase-2 in bladder epithelial cells was significantly increased in the carcinogenesis group compared with the control group. In contrast, in ergosterol-treated rats, these increases were significantly suppressed. Ergosterol did not affect the plasma testosterone concentration or the binding of dihydrotestosterone to androgen receptor (AR). The mRNA expression levels of 5α-reductase type 2 and AR were higher in the carcinogenesis group than in the control group but were significantly decreased by ergosterol administration. These results suggest that ergosterol inhibits bladder carcinogenesis by modulating various aspects of the cell cycle, inflammation-related signaling, and androgen signaling. Future clinical application of the preventive effect of ergosterol on bladder carcinogenesis is expected.

Subnormal IgG1 or IgG3 levels occurred in 30% of hemochromatosis probands with HFE p.C282Y homozygosity and were concordant in HLA-identical siblings. We sought to identify factors associated with IgG subclasses in Alabama probands with p.C282Y homozygosity evaluated for 500 kb microhaplotypes AAT and GGG defined by SNPs in chromosome 6p genes PGBD1, ZNF193, and ZNF165. In regressions on IgG subclasses, we used: age; sex; GGG (dichotomous); iron removed to achieve depletion; CD8+ T-lymphocytes; and other IgG subclasses. Among 49 probands, AAT and GGG occurred in 95.9% and 16.3%, respectively. Thirteen probands (26.5%) had subnormal IgG1; 11 probands (22.4%) had subnormal IgG3. Mean IgG3 was higher in probands with than without GGG (75 mg/dL [95% confidence interval 63, 89] vs. 58 mg/dL [49, 71], respectively; p = 0.0321). Regression on IgG3 revealed: GGG positivity (p = 0.0106); and IgG1 (p = 0.0015). In a replication cohort of 22 Portugal probands with p.C282Y homozygosity, mean IgG3 was higher in probands with than without GGG (46 ± 16 vs. 31 ± 12 mg/dL, respectively; p = 0.0410). We conclude that mean IgG3 levels are higher in hemochromatosis probands with p.C282Y homozygosity with chromosome 6p microhaplotype GGG than in probands homozygous for microhaplotype AAT.

Osteoporosis is characterized by systemic microarchitecture impairment and bone loss, which ultimately lead to fragility fractures. This disease is most common in older people, especially in postmenopausal women. Cancellous bone is affected by osteoporosis earlier than cortical bone, and DNA methylation microarray analysis of the hip cancellous bone of patients with osteoarthritis revealed differential methylation. In view of the important role of cancellous bone in bone development, we examined genome-wide DNA methylation profiles in the cancellous bone from patients with postmenopausal osteoporosis versus healthy postmenopausal women using Illumina 850K methylation microarray analysis. Under a threshold of P < 0.05, we obtained a total of 8973 differentially methylated genes, such as SOX6, ACE, SYK and TGFB3. Under a threshold of P < 0.05 and |△β| > 0.2, a total of 17 and 34 key differentially methylated genes were further identified at the promoter region and cytosine- phosphate- guanine (CpG) islands (such as PRKCZ, GNA11 and COL4A1), respectively. PLEKHA2, PLEKHB1, PNPLA7, SCD, MGST3 and TSNAX were the most common differentially methylated genes at both the promoter region and CpG islands. Five important signaling pathways, including the calcium signaling pathway, the cyclic guanosine phospho-protein kinase G (cGMP-PKG) signaling pathway, endocytosis, the Rap1 signaling pathway and the AMPK signaling pathway were identified. Our study may be suitable as a basis for exploring the mechanisms underlying osteoporosis in postmenopausal women.

An efficient route to various vancoresmycin-type tetramic acids has been developed. The modular route is based on an effective Fries-type rearrangement to introduce various appending acetyl residues. The minimum inhibitory concentration (MIC) values of the new tetramic acids against Staphylococcus aureus and Escherichia coli were determined, revealing that three of the new compounds exhibit antimicrobial activity against S. aureus. These bioactive compounds were structurally most closely related to the authentic vancoresmycin building block. Additionally, the compounds induced a lial-lux bioreporter, which responds to cell wall stress induced by antibiotics that interfere with the lipid II biosynthesis cycle. These data suggest the tetramic acid moiety to be a part of the vancoresmycin pharmacophore.

Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.

Macroautophagy/autophagy is a membrane-mediated intracellular degradation pathway, through which bulky cytoplasmic content is digested in lysosomes. How the autophagy initiation and maturation steps are regulated is not clear. In this study, we found an E3 ubiquitin ligase complex, linear ubiquitin chain assembly complex (LUBAC) and a deubiquitinating enzyme (DUB) OTULIN localize to the phagophore area to control autophagy initiation and maturation. LUBAC key component RNF31/HOIP translocates to the LC3 puncta area when autophagy is induced. <i>RNF31</i> knockdown inhibits autophagy initiation, and cells are more sensitive to bacterial infection. <i>OTULIN</i> knockdown, however, promotes autophagy initiation but blocks autophagy maturation. In <i>OTULIN</i> knockdown cells, excessive ubiquitinated ATG13 protein was recruited to the phagophore for prolonged expansion, and therefore inhibits autophagosome maturation. Together, our study provides evidence that LUBAC and OTULIN cooperatively regulate autophagy initiation and autophagosome maturation by mediating the linear ubiquitination and the stabilization of ATG13.<b>Abbreviations:</b> ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; CUL1-FBXL20: cullin 1-F-box and leucine rich repeat protein 20; CUL3-KLHL20: cullin 3-kelch like family member 20; CUL4-AMBRA1: cullin 4-autophagy and beclin 1 regulator 1; CYLD: CYLD lysine 63 deubiquitinase; DAPI: 4',6-diamidino-2-phenylindole; DUB: deubiquitinating enzyme; EBSS: Earle's Balanced Salt Solution; GFP: green fluorescent protein; GST: glutathione S-transferase; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; LUBAC: linear ubiquitin chain assembly complex; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3B; MIM: MIT-interacting motif; mRFP: monomeric red fluorescent protein; NEDD4: NEDD4 E3 ubiquitin protein ligase; NFKB: NF-kappaB complex; OPTN: optineurin; OTULIN: OTU deubiquitinase with linear linkage specificity; PIK3C3/Vps34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns: phosphatidylinositol; PtdIns3K: class III phosphatidylinositol 3-kinase complex; PtdIns3P: phosphatidylinositol 3-phosphate; RBCK1/HOIL1: RANBP2-type and C3HC4-type zinc finger containing 1; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RIPK1: receptor interacting serine/threonine kinase 1; RNF216: ring finger protein 216; RNF31/HOIP: ring finger protein 31; RT-PCR: reverse transcriptase polymerase chain reaction; S. Typhimurium: <i>Salmonella enterica</i> serovar Typhimurium; SHARPIN: SHANK associated RH domain interactor; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING: stimulator of interferon response cGAMP interactor 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TNF/TNF-alpha: tumor necrosis factor; TNFAIP3/A20: TNF alpha induced protein 3; TRAF6: TNF receptor associated factor 6; TRIM32: tripartite motif containing 32; UBAN: ubiquitin binding in TNIP/ABIN and IKBKG/NEMO proteins; ULK1/2: unc-51 like autophagy activating kinase 1/2; USP: ubiquitin specific peptidase; UVRAG: UV radiation resistance associated; VCPIP1: valosin containing protein interacting protein 1; WIPI2: WD repeat domain, phosphoinositide interacting protein 2; ZBTB16-CUL3-RBX1: zinc finger and BTB domain containing protein 16-cullin 3-ring-box 1; ZRANB1: zinc finger RANBP2-type containing 1.

<h4>Objective</h4>T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease resulting from the accumulation of genetic changes that affect the development of T-cells. The precise role of lymphoid enhancer-binding factor 1 (<i>LEF1</i>) in T-ALL has been controversial since both overexpression and inactivating <i>LEF1</i> mutations have been reported to date. Here, we investigate the potential gene targets of <i>LEF1</i> in the Jurkat human T-cell leukemia cell line.<h4>Materials and methods</h4>We used small interfering RNA (siRNA) technology to knock down <i>LEF1</i> in Jurkat cells and then compared the gene expression levels in the <i>LEF1</i> knockdown cells with non-targeting siRNA-transfected and non-transfected cells by employing microarray analysis.<h4>Results</h4>We identified <i>DHRS2</i>, a tumor suppressor gene, as the most significantly downregulated gene in <i>LEF1</i> knockdown cells, and we further confirmed its downregulation by real-time quantitative polymerase chain reaction (qRT-PCR) in mRNA and at protein level by western blotting.<h4>Conclusion</h4>Our results revealed that <i>DHRS2</i> is positively regulated by <i>LEF1</i> in Jurkat cells, which indicates the capability of <i>LEF1</i> as a tumor suppressor and, together with previous reports, suggests that <i>LEF1</i> exhibits a regulatory role in T-ALL via not only its oncogenic targets but also tumor suppressor genes.

While pregnancy may accelerate glioblastoma multiforme (GBM) growth, parity and progesterone (P4) containing treatments (ie, hormone replacement therapy) reduce the risk of GBM development. In parallel, low and high doses of P4 exert stimulating and inhibitory actions on GBM growth, respectively. The mechanisms behind the high-dose P4-suppression of GBM growth is unknown. In the present study, we assessed the changes in growth and proteomic profiles when high-dose P4 (100 and 300 µM) was administered in human U87 and A172 GBM cell lines. The xCELLigence system was used to examine cell growth when different concentrations of P4 (20, 50, 100, and 300 µM) was administered. The protein profiles were determined by two-dimensional gel electrophoresis in both cell lines when 100 and 300 µM P4 were administered. Finally, the pathways enriched by the differentially expressed proteins were assessed using bioinformatic tools. Increasing doses of P4 blocked the growth of both GBM cells. We identified 26 and 51 differentially expressed proteins (fc > 2) in A172 and U87 cell lines treated with P4, respectively. Only the pro-tumorigenic mitochondrial ornithine aminotransferase and anti-apoptotic mitochondrial 60 kDa heat shock protein were downregulated in A172 cell line and U87 cell line when treated with P4, respectively. Detoxification of reactive oxygen species, cellular response to stress, glucose metabolism, and immunity-related proteins were altered in P4-treated GBM cell lines. The paradox on the effect of low and high doses of P4 on GBM growth is gaining attention. The mechanism related to the high dose of P4 on GBM growth can be explained by the alterations in detoxification mechanisms, stress, and immune response and glucose metabolism. P4 suppresses GBM growth and as it is nontoxic in comparison to classical chemotherapeutics, it can be used as a new strategy in GBM treatment in the future.

Genome-wide mapping of chromatin interactions at high resolution remains experimentally and computationally challenging. Here we used a low-input "easy Hi-C" protocol to map the 3D genome architecture in human neurogenesis and brain tissues and also demonstrated that a rigorous Hi-C bias-correction pipeline (HiCorr) can significantly improve the sensitivity and robustness of Hi-C loop identification at sub-TAD level, especially the enhancer-promoter (E-P) interactions. We used HiCorr to compare the high-resolution maps of chromatin interactions from 10 tissue or cell types with a focus on neurogenesis and brain tissues. We found that dynamic chromatin loops are better hallmarks for cellular differentiation than compartment switching. HiCorr allowed direct observation of cell-type- and differentiation-specific E-P aggregates spanning large neighborhoods, suggesting a mechanism that stabilizes enhancer contacts during development. Interestingly, we concluded that Hi-C loop outperforms eQTL in explaining neurological GWAS results, revealing a unique value of high-resolution 3D genome maps in elucidating the disease etiology.

Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. Here, we use transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, we characterize the in vivo function of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. Our study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo.

MicroRNAs (miRNAs), a family of non-coding RNAs, have recently gained a considerable attention in neuropsychiatric disorders. Being a pleiotropic modulator of target gene(s), miRNA has been recognized as central to downstream gene regulatory networks. In the recent past, reports have suggested their role in changing the epigenetic landscape in brain of subjects with major depressive disorder (MDD). Anterior cingulate cortex (ACC) is a brain area implicated in several complex cognitive functions, such as impulse control, emotion, and decision-making and is associated with psychopathology associated with mood regulation. In this study, we examined whether MDD is associated with altered miRNA transcriptome in ACC and whether altered miRNA landscape is associated with modifications in specific gene network(s) at the functional level. Using next generation sequencing (NGS), it was observed that 117 miRNAs (4.61%) were significantly upregulated and 54 (2.13%) were downregulated in MDD subjects (n = 22) compared with non-psychiatric controls (n = 25). Using 24 most significantly upregulated miRNAs in the MDD group, we determined functional enrichment of target genes and found them to be associated with long-term potentiation, neurotrophin signaling, and axon guidance. Intra- and inter-cluster similarities of enriched terms based on overrepresented gene list showed neurobiological functions associated with neuronal growth and survival. Web centric parameters and ontology enrichment functions identified two major domains related to phosphatidyl signaling, GTPase signaling, neuronal migration, and neurotrophin signaling. Our findings of altered miRNA landscape along with a shift in targetome relate to previously reported morphometric changes and neuronal atrophy in ACC of MDD subjects.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of ²⁶Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)₂: (²⁰RRWQWRMKKLG³⁰)₂-K-<i>Ahx</i> with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)₂ analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC₅₀ values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

In silico models to predict which tumors will respond to a given drug are necessary for Precision Oncology. However, predictive models are only available for a handful of cases (each case being a given drug acting on tumors of a specific cancer type). A way to generate predictive models for the remaining cases is with suitable machine learning algorithms that are yet to be applied to existing in vitro pharmacogenomics datasets. Here, we apply XGBoost integrated with a stringent feature selection approach, which is an algorithm that is advantageous for these high-dimensional problems. Thus, we identified and validated 118 predictive models for 62 drugs across five cancer types by exploiting four molecular profiles (sequence mutations, copy-number alterations, gene expression, and DNA methylation). Predictive models were found in each cancer type and with every molecular profile. On average, no omics profile or cancer type obtained models with higher predictive accuracy than the rest. However, within a given cancer type, some molecular profiles were overrepresented among predictive models. For instance, CNA profiles were predictive in breast invasive carcinoma (BRCA) cell lines, but not in small cell lung cancer (SCLC) cell lines where gene expression (GEX) and DNA methylation profiles were the most predictive. Lastly, we identified the best XGBoost model per cancer type and analyzed their selected features. For each model, some of the genes in the selected list had already been found to be individually linked to the response to that drug, providing additional evidence of the usefulness of these models and the merits of the feature selection scheme.

Cleavage and polyadenylation are essential processes that can impact many aspects of mRNA fate. Most eukaryotic genes have alternative polyadenylation (APA) events. While the heterogeneity of mRNA polyadenylation isoform choice has been studied in specific tissues, less attention has been paid to the neuronal heterogeneity of APA selection at single-nucleus resolution. APA is highly controlled during development and neuronal activation, however, to what extent APA events vary in a specific neuronal cell population and the regulatory mechanisms are still unclear. In this paper, we investigated dynamic APA usage in different cell types using snRNA-seq data of 1424 human brain cells generated by single-cell 3' RNA sequencing. We found that distal APA sites are not only favored by global neuronal cells, but that their usage also varies between the principal types of neuronal cell populations (excitatory neurons and inhibitory neurons). A motif analysis and a gene functional analysis indicated the enrichment of RNA-binding protein (RBP) binding sites and neuronal functions for the set of genes with neuron-enhanced distal PAS usage. Our results revealed the extensive involvement of APA regulation in neuronal populations at the single-nucleus level, providing new insights into roles for APA in specific neuronal cell populations, as well as utility in future functional studies.

<h4>Background</h4>Exocrine pancreatic insufficiency results from the destruction of the pancreatic parenchyma and is diagnosed by using direct or indirect tests, both of which have shortcomings. Chronic pancreatitis is the most frequent cause of this pathology in adults.<h4>Methods</h4>Patients meeting radiological or histological diagnostic criteria of chronic pancreatitis are enrolled and the stool elastase test is conducted, considering fecal elastase levels >200 µg/g to represent normal pancreatic function, and levels <200 μg/g to indicate the presence of exocrine pancreatic insufficiency. Additionally, we determine the body mass index of the patients and study their nutritional status and main biochemical and hematological variables, including their glucose and hemoglobin A1c (HbA1c) levels.<h4>Results</h4>Exocrine pancreatic insufficiency is detected in 60% of the patients. Among these, 83.3% are severe cases, and 72% of the latter also are diagnosed with endocrine pancreatic insufficiency (diabetes mellitus). During the nutritional status study, HbA1c levels are significantly higher, and magnesium and prealbumin levels are significantly lower in patients with exocrine pancreatic insufficiency than in those without this disease.<h4>Conclusions</h4>Exocrine and endocrine pancreatic insufficiency are highly prevalent among patients with chronic pancreatitis and an early diagnosis of these diseases is vital to improve the clinical management of these patients and reduce their risk of mortality.

The synergy between photocatalysis and continuous flow chemical reactors has shifted the paradigms of photochemistry, opening new avenues of research with safer and scalable processes that can be readily implemented in academia and industry. Current state-of-the-art photocatalysts are homogeneous transition metal complexes that have favourable photophysical properties, wide electrochemical redox potentials, and photostability. However, these photocatalysts present serious drawbacks, such as toxicity, limited availability, and the overall cost of rare transition metal elements. This reduces their long-term viability, especially at an industrial scale. Heterogeneous photocatalysts (HPCats) are an attractive alternative, as the requirement for the separation and purification is largely removed, but typically at the cost of efficiency. Flow chemical reactors can, to a large extent, mitigate the loss in efficiency through reactor designs that enhance mass transport and irradiation. Herein, we review some important developments of heterogeneous photocatalytic materials and their application in flow reactors for sustainable organic synthesis. Further, the application of continuous flow heterogeneous photocatalysis in environmental remediation is briefly discussed to present some interesting reactor designs that could be implemented to enhance organic synthesis.

Checkpoint kinase 1 (CHK1) is a serine/threonine-protein kinase that is involved in cell cycle regulation in eukaryotes. Inhibition of CHK1 is thus considered as a promising approach in cancer therapy. In this study, the fast pulling of ligand (FPL) process was applied to predict the relative binding affinities of CHK1 inhibitors using non-equilibrium molecular dynamics (MD) simulations. The work of external harmonic forces to pull the ligand out of the binding cavity strongly correlated with the experimental binding affinity of CHK1 inhibitors with the correlation coefficient of R = -0.88 and an overall root mean square error (RMSE) of 0.99 kcal/mol. The data indicate that the FPL method is highly accurate in predicting the relative binding free energies of CHK1 inhibitors with an affordable CPU time. A new set of molecules were designed based on the molecular modeling of interactions between the known inhibitor and CHK1 as inhibitory candidates. Molecular docking and FPL results exhibited that the binding affinities of developed ligands were similar to the known inhibitor in interaction with the catalytic site of CHK1, producing very potential CHK1 inhibitors of that the inhibitory activities should be further evaluated in vitro.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.

<h4>Introduction</h4>The fundamental theory of traditional Chinese medicine (TCM) implies that when different diseases have the same pathogen, the syndromes of these individual diseases will be the same. "Treating different diseases with the same method" is a TCM principle suggesting that when different diseases have similar pathological changes during different stages of their development, the same method of treatment can be applied. Our study aims to analyze the concept "treating different diseases with the same method" from a molecular perspective, in order to clarify its biological basis and to objectively standardize future TCM syndrome research.<h4>Objective</h4>The TCM syndromes Qi deficiency and blood stasis have similar pathogenesis in relation to coronary heart disease (CHD) and stroke. We aim to use big data technology and complex network theory to mine the genes specifically relevant to these TCM syndromes. This study aims to explore the correlation between the biological indicators of CHD and stroke from a scientific perspective.<h4>Methods</h4>Mining the relevant neuroendocrine-immune (NEI) genes by means of gene entity recognition, complex network construction, network integration, and decomposition to categorize relevant syndrome terms and establish a digital dictionary of gene specifically related to individual diseases. We analyzed the biological basis of "treating different diseases with the same method" from a molecular level using the TCMIP v2.0 platform in order to categorize the TCM syndromes most relevant to CHD and stroke.<h4>Results</h4>We found 46 genes were involved in the TCM syndromes of Qi deficiency and blood stasis of CHD and stroke. The same genes and their molecular mechanism also appeared to be in close relation to inflammatory response, apoptosis, and proliferation.<h4>Conclusion</h4>By using information extraction and complex network technology, we discovered the biological indicators of the TCM syndromes Qi deficiency and blood stasis of CHD and stroke. In the era of big data, our results can provide a new method for the researchers of TCM syndrome differentiation, as well as an effective and specific methodology for standardization of TCM.

<b>Background:</b> Osteosarcoma is a solid tumor common in the musculoskeletal system. The DEAD-box helicase (DDX) families play an important role in tumor genesis and proliferation. <b>Objective:</b>To screen potential molecular targets in osteosarcoma and elucidate its relationship with DDX56. <b>Methods:</b> We employed the Gene Expression Omnibus and The Cancer Genome Atlas datasets for preliminary screening. DDX56 expression was measured by RT-qPCR in three osteosarcoma cell lines. Biological roles of DDX56 were explored by Gene ontology, Kyoto Encyclopedia of Genes and Genomes and Ingenuity Pathway Analysis. Cell proliferation, cycle, and apoptosis assays were performed using Lentivirus™ knockdown technique. <b>Results:</b> It was found that DDX56 expression was regularly upregulated in osteosarcoma tissue and cell lines, while DDX56 knockdown inhibited cell proliferation and promoted cell apoptosis. <b>Conclusions:</b> The findings suggest DDX56 as a potential therapeutic target for the treatment of osteosarcoma.

Yes-associated protein (YAP) is a key effector downstream of the Hippo signaling pathway and plays an important role in the development of the physiology and pathology of the central nervous system (CNS), especially regulating cell proliferation, differentiation, migration, and apoptosis. However, the roles and underlying mechanisms of YAP in CNS diseases are still puzzling. Here, this review will systematically and comprehensively summarize the biological feature, pathological role, and underlying mechanisms of YAP in normal and pathologic CNS, which aims to provide insights into the potential molecular targets and new therapeutic strategies for CNS diseases.

Ferroptosis is a new mode of cell death that is characterized by the excessive accumulation of iron and lipid peroxides. It has unique morphological changes and disparate biochemical features and plays an intricate role in many pathophysiological processes. A great deal of researches confirms that ferroptosis can be regulated by numerous molecules through different mechanisms, supporting great potentials for novel pharmacological therapeutics. Recently, several studies reveal that ferroptosis is also closely associated with the initiation and development of respiratory disease. Understanding the specific mechanism, the molecular trait of ferroptosis and their relationship with pulmonary disease could provide significant references regarding effective treatment of these obstinate disease.

<i>Background</i>: Abnormally low or high blood iron levels are common health conditions worldwide and can seriously affect an individual's overall well-being. A low-cost point-of-care technology that measures blood iron markers with a goal of both preventing and treating iron-related disorders represents a significant advancement in medical care delivery systems. <i>Methods:</i> A novel assay equipped with an accurate, storable, and robust dry sensor strip, as well as a smartphone mount and (iPhone) app is used to measure total iron in human serum. The sensor strip has a vertical flow design and is based on an optimized chemical reaction. The reaction strips iron ions from blood-transport proteins, reduces Fe(III) to Fe(II), and chelates Fe(II) with ferene, with the change indicated by a blue color on the strip. The smartphone mount is robust and controls the light source of the color reading App, which is calibrated to obtain output iron concentration results. The real serum samples are then used to assess iron concentrations from the new assay, and validated through intra-laboratory and inter-laboratory experiments. The intra-laboratory validation uses an optimized iron detection assay with multi-well plate spectrophotometry. The inter-laboratory validation method is performed in a commercial testing facility (LabCorp). <i>Results</i>: The novel assay with the dry sensor strip and smartphone mount, and App is seen to be sensitive to iron detection with a dynamic range of 50 - [Formula: see text]/dL, sensitivity of 0.00049 a.u/[Formula: see text]/dL, coefficient of variation (CV) of 10.5%, and an estimated detection limit of [Formula: see text]/dL These analytical specifications are useful for predicting iron deficiency and overloads. The optimized reference method has a sensitivity of 0.00093 a.u/[Formula: see text]/dL and CV of 2.2%. The correlation of serum iron concentrations (N = 20) between the optimized reference method and the novel assay renders a slope of 0.95, and a regression coefficient of 0.98, suggesting that the new assay is accurate. Last, a spectrophotometric study of the iron detection reaction kinetics is seen to reveal the reaction order for iron and chelating agent. <i>Conclusion:</i> The new assay is able to provide accurate results in intra- and inter- laboraty validations, and has promising features of both mobility and low-cost manufacturing suitable for global healthcare settings.

Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage-specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of <i>Tet1</i> via short-hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of <i>Tet1</i> KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome-wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte-specific class II binding sites for SOX9, as compared with the tissue-agnostic class I sites. Specifically, we find that SOX9 is unable to bind to <i>Col2a1</i> and <i>Acan</i> after <i>Tet1</i> KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using <i>Tet1</i> ^<i>-/-</i> embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 <i>Tet1</i> ^<i>-/-</i> embryos, loss of SOX9 target gene expression is more modest than upon <i>Tet1</i> KD in vitro<i>.</i> Overall, our data suggest a role for TET1-mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. <i>JBMR Plus</i> published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Chronic pain is highly prevalent worldwide and imparts significant socioeconomic and public health burden and is more prevalent in women than in men. Factors that influence susceptibility and mechanisms of chronic pain development, are not fully understood. To investigate sex differences in chronic pain, we carried out a sex-stratified genome-wide association study of Multisite Chronic Pain (MCP), a derived chronic pain phenotype in UK Biobank. Genetic correlations between MCP in each sex and a range of psychiatric, autoimmune, and anthropometric phenotypes were examined. The relationship between female and male MCP, and chronic widespread pain was investigated using polygenic risk scoring. Expression of sex-specific MCP-associated loci in a range of tissues was examined using GTEx, and separately in neural and non-neural human tissues with assessment for dorsal-root ganglion (DRG) enrichment. For genes enriched for neural-tissue expression the full GTEx database was queried for sex-differential gene expression in CNS regions, and for high expression in sex-specific tissues. Expression in neural mouse tissue was also examined for orthologs of these genes. A total of 123 SNPs at five independent loci were significantly associated with MCP in men. In women, a total of 286 genome-wide significant SNPs at ten independent loci were discovered. Meta-analysis of sex-stratified GWAS output found 87 independent SNPs to be significantly associated with MCP. We found sex-specific MCP-associated genes, with 31 genes and 37 genes associated with female and male MCP respectively and one gene associated with MCP in both sexes ( DCC ). We found evidence for sex-specific pleiotropy and risk for MCP was found to be associated with chronic widespread pain in a sex-differential manner. Male and female MCP were highly genetically correlated, but at r g significantly less than 1. All 37 male MCP-associated genes and all but one of 31 female MCP-associated genes were found to be expressed in the DRG, and many showed elevated expression in sex-specific tissues. Overall, findings indicate sex differences in chronic pain at the SNP, gene and transcriptomic level, and highlight possible sex-specific pleiotropy for MCP. Results support the proposition of a strong nervous-system component to chronic pain in both sexes, emphasise the importance of the DRG, and indicate specific loci which may play a specialised role in nociception.

<h4>Background</h4>Immune challenge is known to increase heat stroke risk, although the mechanism of this increased risk is unclear.<h4>Objectives</h4>We sought to understand the effect of immune challenge on heat stroke pathology.<h4>Patients/methods</h4>Using a mouse model of classic heat stroke, we examined the impact of prior viral or bacterial infection on hematological aspects of recovery. Mice were exposed to heat either 48 or 72 hours following polyinosinic:polycytidylic acid (poly I:C) or lipopolysaccharide injection, time points when symptoms of illness (fever, lethargy, anorexia) were minimized or completely absent.<h4>Results</h4>Employing multivariate supervised machine learning to identify patterns of molecular and cellular markers associated with heat stroke, we found that prior viral infection simulated with poly I:C injection resulted in heat stroke presenting with high levels of factors indicating coagulopathy. Despite a decreased number of platelets in the blood, platelets are large and non-uniform in size, suggesting younger, more active platelets. Levels of D-dimer and soluble thrombomodulin were increased in more severe heat stroke, and in cases of the highest level of organ damage markers D-dimer levels dropped, indicating potential fibrinolysis-resistant thrombosis. Genes corresponding to immune response, coagulation, hypoxia, and vessel repair were up-regulated in kidneys of heat-challenged animals; these correlated with both viral treatment and distal organ damage while appearing before discernible tissue damage to the kidney itself.<h4>Conclusions</h4>Heat stroke-induced coagulopathy may be a driving mechanistic force in heat stroke pathology, especially when exacerbated by prior infection. Coagulation markers may serve as accessible biomarkers for heat stroke severity and therapeutic strategies.

Adherent-invasive <i>E. coli</i> (AIEC), which abnormally colonize the intestinal mucosa of Crohn's disease (CD) patients, are able to adhere to and invade intestinal epithelial cells (IECs), survive and replicate within macrophages and induce a pro-inflammatory response. AIEC infection of IECs induces secretion of exosomes that increase AIEC replication in exosome-receiving IECs and macrophages. Here, we investigated the mechanism underlying the increased AIEC replication in cells receiving exosomes from AIEC-infected cells. Exosomes released by uninfected human intestinal epithelial T84 cells (Exo-uninfected) or by T84 cells infected with the clinical AIEC LF82 strain (Exo-LF82), the nonpathogenic <i>E. coli</i> K12 strain (Exo-K12) or the commensal <i>E. coli</i> HS strain (Exo-HS) were purified and used to stimulate T84 cells. Stimulation of T84 cells with Exo-LF82 inhibited autophagy compared with Exo-uninfected, Exo-K12 and Exo-HS. qRT-PCR analysis revealed increased levels of miR-30c and miR-130a in Exo-LF82 compared to Exo-uninfected, Exo-K12 and Exo-HS. These miRNAs were transferred <i>via</i> exosomes to recipient cells, in which they targeted and inhibited ATG5 and ATG16L1 expression and thereby autophagy response, thus favoring AIEC intracellular replication. Inhibition of these miRNAs in exosome-donor cells infected with AIEC LF82 abolished the increase in miR-30c and miR-130a levels in the released Exo-LF82 and in Exo-LF82-receiving cells, thus suppressing the inhibitory effect of Exo-LF82 on ATG5 and ATG16L1 expression and on autophagy-mediated AIEC clearance in Exo-LF82-receiving cells. Our study shows that upon AIEC infection, IECs secrete exosomes that can transfer specific miRNAs to recipient IECs, inhibiting autophagy-mediated clearance of intracellular AIEC.

<h4>Background</h4>The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and resulting COVID-19 pandemic present important diagnostic challenges. Several diagnostic strategies are available to identify current infection, rule out infection, identify people in need of care escalation, or to test for past infection and immune response. Serology tests to detect the presence of antibodies to SARS-CoV-2 aim to identify previous SARS-CoV-2 infection, and may help to confirm the presence of current infection.<h4>Objectives</h4>To assess the diagnostic accuracy of antibody tests to determine if a person presenting in the community or in primary or secondary care has SARS-CoV-2 infection, or has previously had SARS-CoV-2 infection, and the accuracy of antibody tests for use in seroprevalence surveys.<h4>Search methods</h4>We undertook electronic searches in the Cochrane COVID-19 Study Register and the COVID-19 Living Evidence Database from the University of Bern, which is updated daily with published articles from PubMed and Embase and with preprints from medRxiv and bioRxiv. In addition, we checked repositories of COVID-19 publications. We did not apply any language restrictions. We conducted searches for this review iteration up to 27 April 2020.<h4>Selection criteria</h4>We included test accuracy studies of any design that evaluated antibody tests (including enzyme-linked immunosorbent assays, chemiluminescence immunoassays, and lateral flow assays) in people suspected of current or previous SARS-CoV-2 infection, or where tests were used to screen for infection. We also included studies of people either known to have, or not to have SARS-CoV-2 infection. We included all reference standards to define the presence or absence of SARS-CoV-2 (including reverse transcription polymerase chain reaction tests (RT-PCR) and clinical diagnostic criteria).<h4>Data collection and analysis</h4>We assessed possible bias and applicability of the studies using the QUADAS-2 tool. We extracted 2x2 contingency table data and present sensitivity and specificity for each antibody (or combination of antibodies) using paired forest plots. We pooled data using random-effects logistic regression where appropriate, stratifying by time since post-symptom onset. We tabulated available data by test manufacturer. We have presented uncertainty in estimates of sensitivity and specificity using 95% confidence intervals (CIs).<h4>Main results</h4>We included 57 publications reporting on a total of 54 study cohorts with 15,976 samples, of which 8526 were from cases of SARS-CoV-2 infection. Studies were conducted in Asia (n = 38), Europe (n = 15), and the USA and China (n = 1). We identified data from 25 commercial tests and numerous in-house assays, a small fraction of the 279 antibody assays listed by the Foundation for Innovative Diagnostics. More than half (n = 28) of the studies included were only available as preprints. We had concerns about risk of bias and applicability. Common issues were use of multi-group designs (n = 29), inclusion of only COVID-19 cases (n = 19), lack of blinding of the index test (n = 49) and reference standard (n = 29), differential verification (n = 22), and the lack of clarity about participant numbers, characteristics and study exclusions (n = 47). Most studies (n = 44) only included people hospitalised due to suspected or confirmed COVID-19 infection. There were no studies exclusively in asymptomatic participants. Two-thirds of the studies (n = 33) defined COVID-19 cases based on RT-PCR results alone, ignoring the potential for false-negative RT-PCR results. We observed evidence of selective publication of study findings through omission of the identity of tests (n = 5). We observed substantial heterogeneity in sensitivities of IgA, IgM and IgG antibodies, or combinations thereof, for results aggregated across different time periods post-symptom onset (range 0% to 100% for all target antibodies). We thus based the main results of the review on the 38 studies that stratified results by time since symptom onset. The numbers of individuals contributing data within each study each week are small and are usually not based on tracking the same groups of patients over time. Pooled results for IgG, IgM, IgA, total antibodies and IgG/IgM all showed low sensitivity during the first week since onset of symptoms (all less than 30.1%), rising in the second week and reaching their highest values in the third week. The combination of IgG/IgM had a sensitivity of 30.1% (95% CI 21.4 to 40.7) for 1 to 7 days, 72.2% (95% CI 63.5 to 79.5) for 8 to 14 days, 91.4% (95% CI 87.0 to 94.4) for 15 to 21 days. Estimates of accuracy beyond three weeks are based on smaller sample sizes and fewer studies. For 21 to 35 days, pooled sensitivities for IgG/IgM were 96.0% (95% CI 90.6 to 98.3). There are insufficient studies to estimate sensitivity of tests beyond 35 days post-symptom onset. Summary specificities (provided in 35 studies) exceeded 98% for all target antibodies with confidence intervals no more than 2 percentage points wide. False-positive results were more common where COVID-19 had been suspected and ruled out, but numbers were small and the difference was within the range expected by chance. Assuming a prevalence of 50%, a value considered possible in healthcare workers who have suffered respiratory symptoms, we would anticipate that 43 (28 to 65) would be missed and 7 (3 to 14) would be falsely positive in 1000 people undergoing IgG/IgM testing at days 15 to 21 post-symptom onset. At a prevalence of 20%, a likely value in surveys in high-risk settings, 17 (11 to 26) would be missed per 1000 people tested and 10 (5 to 22) would be falsely positive. At a lower prevalence of 5%, a likely value in national surveys, 4 (3 to 7) would be missed per 1000 tested, and 12 (6 to 27) would be falsely positive. Analyses showed small differences in sensitivity between assay type, but methodological concerns and sparse data prevent comparisons between test brands.<h4>Authors' conclusions</h4>The sensitivity of antibody tests is too low in the first week since symptom onset to have a primary role for the diagnosis of COVID-19, but they may still have a role complementing other testing in individuals presenting later, when RT-PCR tests are negative, or are not done. Antibody tests are likely to have a useful role for detecting previous SARS-CoV-2 infection if used 15 or more days after the onset of symptoms. However, the duration of antibody rises is currently unknown, and we found very little data beyond 35 days post-symptom onset. We are therefore uncertain about the utility of these tests for seroprevalence surveys for public health management purposes. Concerns about high risk of bias and applicability make it likely that the accuracy of tests when used in clinical care will be lower than reported in the included studies. Sensitivity has mainly been evaluated in hospitalised patients, so it is unclear whether the tests are able to detect lower antibody levels likely seen with milder and asymptomatic COVID-19 disease. The design, execution and reporting of studies of the accuracy of COVID-19 tests requires considerable improvement. Studies must report data on sensitivity disaggregated by time since onset of symptoms. COVID-19-positive cases who are RT-PCR-negative should be included as well as those confirmed RT-PCR, in accordance with the World Health Organization (WHO) and China National Health Commission of the People's Republic of China (CDC) case definitions. We were only able to obtain data from a small proportion of available tests, and action is needed to ensure that all results of test evaluations are available in the public domain to prevent selective reporting. This is a fast-moving field and we plan ongoing updates of this living systematic review.

Previous research regarding Holstein cows has mainly focused on increasing milk yield. However, in order to maximize the economical profits of Holstein cattle farming, it is necessary to fully take advantage of Holstein bulls to produce high-grade beef. The present study aims to investigate different transcriptomic profiling of Holstein bulls and steers, via high-throughput RNA-sequencing (RNA-seq). The growth and beef quality traits of Holstein steers and bulls were characterized via assessment of weight, rib eye area, marbling score, shear force and intramuscular fat percentage of the longissimus lumborum (LL) muscle. The results indicated that castration improved the meat quality, yet reduced the meat yield. Subsequently, RNA-seq of the LL muscle from Holstein steers and bulls revealed a total of 56 differentially expressed genes (DEGs). We performed the functional enrichment analysis in Gene Ontology (GO) annotations of the DEGs using GOseq R package software and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using KOBAS tool. Through the integrated analysis of DEGs with reported QTLs and SNPs, seven promising candidate genes potentially affecting the beef quality of LL muscle following castration were discovered, including muscle structural protein coding genes (MYH1, MYH4, MYH10) and functional protein coding genes (GADL1, CYP2R1, EEPD1, SHISA3). Among them, MYH10, GADL1, CYP2R1, EEPD1 and SHISA3 were novel candidate genes associated with beef quality traits. Notably, EEPD1 was associated with both meat quality and reproduction traits, thus indicating its overlapping role in responding to hormone change, and subsequently inducing beef quality improvement. Our findings provide a complete dataset of gene expression profile of LL in Holstein bulls and steers, and will aid in understanding how castration influence meat yield and quality.

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC₅₀ values were found (in micromolars): >1000 (Ca_V2.3), 957 (Ca_V1.3), 656 (Ca_V1.2), 219 (Ca_V3.2), and 132 (Ca_V3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca²⁺ channels and a modest upregulation of Ca_V1.3 and Ca_V2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning Ca_V3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC₅₀ value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (<i>R</i>)-MDIMP [methyl (<i>R</i>)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca²⁺ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

<h4>Background</h4>Hepcidin and hemochromatosis (HFE) are iron regulatory proteins that are encoded by HAMP and HFE genes. Mutation in either HAMP gene or HFE gene causes Hepcidin protein deficiency that can lead to iron overload in beta thalassemia patients. The aim of this research work was to study the presence of G71D mutation of HAMP gene and H63D mutation of HFE gene in beta thalassemia major and minor group to check the association of these mutations with serum ferritin level of beta thalassemia patients.<h4>Methods</h4>The study was conducted on 42 beta thalassemia major and 20 beta thalassemia minor samples along with 20 control samples. The genotyping of both mutations has done by ARM-PCR technique with specific set of primers.<h4>Results</h4>Significant effect of G71D and H63D mutations was observed on serum ferritin level of thalassemia major group. The risk allele of HAMP G71D and HFE H63D was found with high frequency (48% and 49%, respectively) in beta thalassemia major than in control group. High genotypic frequency of HAMP and HFE gene mutation gene mutation was observed in beta thalassemia major than beta thalassemia minor and control group (7% and 9%, respectively).<h4>Conclusion</h4>It can be concluded that both HAMP and HFE gene mutations show high frequency in beta thalassemia major patients and mean significant association between mutations and high serum ferritin level of beta thalassemia major patients but the nonsignificant results of Odd ratios showed that both mutations do not act as major risk factor in beta thalassemia major.

The maturation of the mammalian brain occurs after birth, and this stage of neuronal development is frequently impaired in neurological disorders, such as autism and schizophrenia. However, the mechanisms that regulate postnatal brain maturation are poorly defined. By purifying neuronal subpopulations across brain development in mice, we identify a postnatal switch in the transcriptional regulatory circuits that operates in the maturing mammalian brain. We show that this developmental transition includes the formation of hundreds of cell-type-specific neuronal enhancers that appear to be modulated by neuronal activity. Once selected, these enhancers are active throughout adulthood, suggesting that their formation in early life shapes neuronal identity and regulates mature brain function.

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.

Glial cells play critical roles in the normal development and function of neural circuits, but in many neurodegenerative diseases, they become dysregulated and may contribute to the development of brain pathology. In Huntington's disease (HD), glial cells both lose normal functions and gain neuropathic phenotypes. In addition, cell-autonomous dysfunction elicited by mutant huntingtin (mHTT) expression in specific glial cell types is sufficient to induce both pathology and Huntington's disease-related impairments in motor and cognitive performance, suggesting that these cells may drive the development of certain aspects of Huntington's disease pathogenesis. In support of this imaging studies in pre-symptomatic HD patients and work on mouse models have suggested that glial cell dysfunction occurs at a very early stage of the disease, prior to the onset of motor and cognitive deficits. Furthermore, selectively ablating mHTT from specific glial cells or correcting for HD-induced changes in their transcriptional profile rescues some HD-related phenotypes, demonstrating the potential of targeting these cells for therapeutic intervention. Here we review emerging research focused on understanding the involvement of different glial cell types in specific aspects of HD pathogenesis. This work is providing new insight into how HD impacts biological functions of glial cells in the healthy brain as well as how HD induced dysfunction in these cells might change the way they integrate into biological circuits.

<h4>Background</h4>Individuals with non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), are at increased risk for cardiovascular events, independent of traditional risk factors. Limited data on pro-inflammatory high density lipoprotein (HDL) in NASH exists in the literature. We hypothesized that HDL from individuals with NASH would be more pro-inflammatory than HDL from individuals without NASH.<h4>Methods</h4>Study participants were individuals with obesity who had undergone bariatric surgery with wedge liver biopsy. Using HDL isolated from serum obtained from study participants at the time of surgery, HDL-elicited macrophage cytokine expression (TNF-α, IL-1β, and IL-6) from THP-1 macrophages, HDL-associated receptor expression (ABCA1 and ABCG1) from apolipoprotein E deficient (apo E^-/-) mouse peritoneal macrophages, and isolevuglandin (isoLG) modified HDL were measured.<h4>Results</h4>11 women with NASH and 15 women without NASH were included in the study. Both TNF-α (P = 0.032) and IL-1β (P = 0.029) were significantly more expressed by THP-1 macrophages exposed to HDL from women with NASH compared to women without NASH. ABCA1 and ABCG1 expression by apo E^-/- mouse peritoneal macrophages was not significantly different when exposed to HDL from either women with NASH or women without NASH. IsoLG-modified HDL isolated from the serum of women with NASH trended higher than women without NASH.<h4>Conclusion</h4>Our study suggests a more pro-inflammatory HDL in women with obesity and NASH compared to women with obesity and without NASH.

<h4>Background</h4>Thiazoles, thiazolidinones and azetidinones are highly ranked amongst natural and synthetic heterocyclic derivatives due to their great pharmaceutical potential.<h4>Results</h4>New thiazolidinone and azetidinone class of bioactive agents based on 4-(2,7-dichloro-9<i>H</i>-fluoren-4-yl)thiazole moiety have been successfully synthesized. 4-(2,7-dichloro-9<i>H</i>-fluoren-4-yl)thiazol-2-amine was synthesized and allowed to react with various aryl/heteroaryl aldehydes to afford the corresponding Schiff base intermediates. The target thiazolidinone and azetidinone analogues have derived from Schiff bases by their reactions with thioglycolic acid and chloroacetyl chloride, respectively. The newly synthesized compounds were then evaluated for their antimicrobial activity against some multidrug resistant strains and examined for cytotoxic activity against normal lung fibroblast (WI-38), human lung carcinoma (A549), and human breast carcinoma (MDA-MB-231) cell lines to develop a novel class of fluorene-based bioactive agents. The mode of action and the binding interaction of the synthesized compound with the active sites of dihydrofolate reductase enzyme were well identified by fluorescence-activated cell sorting (FACS) analysis and molecular docking study.<h4>Conclusion</h4>Some of the synthesized compounds showed remarkable activity against A-549 and MDA-MB-231 when compared to Taxol, which was used as a reference drug. 2,7-dichloro-9<i>H</i>-fluorene-based azetidinones are more efficient as antimicrobial and anticancer agents compared to dichloro-9<i>H</i>-fluorene-based thiazolidinones derivatives.

Diabetic retinopathy (DR) is a retinal vascular disorder associated with both type 1 and type 2 diabetes mellitus (DM). It is characterized by specific loss of pericytes, which leads to an augmented blood vessel permeability, and development of new blood vessels (retinal neovascularization). Moreover, stiffening of eye membrane, inflammation, and apoptosis of endothelial cells also lead to damage of the blood-retinal barrier and blindness in most cases unless it's detected and managed early. Hence, this review was intended to assess the potential roles of Netrin-1 and -4 as new/alternative biomarkers and therapeutic options for DR. Netrin-1 and -4 have been the most known ligands and are well known for their role in neural guidance. DR has both neural and vascular components; therefore, biomarkers used for both neural and vascular retinal tissues are potentially important. According to different experimental and clinical studies, as compared to the normal groups, there was a significant increment in both retinal Netrin-1 and -4 mRNA and protein levels in the retinopathy groups. In addition, exogenous supplementation of these proteins is also used as a therapeutic agent for DR.

<h4>Background</h4>Currently, acute myelocytic leukemia (AML) still has a poor prognosis. As a result, gene markers for predicting AML prognosis must be identified through systemic analysis of multi-omics data.<h4>Methods</h4>First of all, the copy number variation (CNV), mutation, RNA-Seq, and single nucleotide polymorphism (SNP) data, as well as those clinical follow-up data, were obtained based on The Cancer Genome Atlas (TCGA) database. Thereafter, all samples (<i>n</i> = 229) were randomized as test set and training set, respectively. Of them, the training set was used to screen for genes related to prognosis, and genes with mutation, SNP or CNV. Then, shrinkage estimate was used for feature selection of all the as-screened genes, to select those stable biomarkers. Eventually, a prognosis model related to those genes was established, and validated within the GEO verification (<i>n</i> = 124 and 72) and test set (<i>n</i> = 127). Moreover, it was compared with the AML prognosis prediction model reported in literature.<h4>Results</h4>Altogether 832 genes related to prognosis, 23 related to copy amplification, 774 associated with copy deletion, and 189 with significant genomic variations were acquired in this study. Later, genes with genomic variations and those related to prognosis were integrated to obtain 38 candidate genes; eventually, a shrinkage estimate was adopted to obtain 10 feature genes (including FAT2, CAMK2A, TCERG1, GDF9, PTGIS, DOC2B, DNTTIP1, PREX1, CRISPLD1 and C22orf42). Further, a signature was established using these 10 genes based on Cox regression analysis, and it served as an independent factor to predict AML prognosis. More importantly, it was able to stratify those external verification, test and training set samples with regard to the risk (<i>P</i> < 0.01). Compared with the prognosis prediction model reported in literature, the model established in this study was advantageous in terms of the prediction performance.<h4>Conclusion</h4>The signature based on 10 genes had been established in this study, which is promising to be used to be a new marker for predicting AML prognosis.

The oncogenic properties of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) have been reported, although the tumor-promoting mechanism remains unclear. We herein report the mechanism underlying colorectal cancer cell progression mediated by hnRNP H1. The growth of colorectal cancer cells was suppressed by hnRNP H1 downregulation. A terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed the anti-apoptotic effect of hnRNP H1 in colorectal cancer cells. An RNA immunoprecipitation assay revealed that hnRNP H1 bound to sphingosine-1-phosphate lyase 1 (SGPL1). Reverse transcription-polymerase chain reaction revealed the high expression of hnRNP H1 mRNA in colorectal cancer cells and Spearman's rank correlation coefficient showed a strong positive correlation between hnRNP H1 mRNA and SGPL1 mRNA. An siRNA of hnRNP H1 decreased SGPL1 mRNA expression in colorectal cancer cells, but not in non-tumorous cells. These findings suggested that hnRNP H1 increased SGPL1 mRNA expression specifically in cancer cells through direct binding. Targeted knockdown of hnRNP H1 or SGPL1 with siRNAs upregulated p53 phosphorylation and p53-associated molecules, resulting in cell growth inhibition, while hnRNP H1 upregulated the mRNA of SGPL1 and inhibited p53 activation, thereby promoting tumor cell growth. This is a novel mechanism underlying colorectal cancer cell progression mediated by hnRNP H1-SGPL1 mRNA stabilization.

Bacterial cellulose (BC) is a promising material for biomedical applications due to its unique properties such as high mechanical strength and biocompatibility. This article describes the microbiological synthesis, modification, and characterization of the obtained BC-nanocomposites originating from symbiotic consortium <i>Medusomyces gisevii</i>. Two BC-modifications have been obtained: BC-Ag and BC-calcium phosphate (BC-Ca₃(PO₄)₂). Structure and physicochemical properties of the BC and its modifications were investigated by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic force microscopy (AFM), and infrared Fourier spectroscopy as well as by measurements of mechanical and water holding/absorbing capacities. Topographic analysis of the surface revealed multicomponent thick fibrils (150-160 nm in diameter and about 15 µm in length) constituted by 50-60 nm nanofibrils weaved into a left-hand helix. Distinctive features of Ca-phosphate-modified BC samples were (a) the presence of 500-700 nm entanglements and (b) inclusions of Ca₃(PO₄)₂ crystals. The samples impregnated with Ag nanoparticles exhibited numerous roundish inclusions, about 110 nm in diameter. The boundaries between the organic and inorganic phases were very distinct in both cases. The Ag-modified samples also showed a prominent waving pattern in the packing of nanofibrils. The obtained BC gel films possessed water-holding capacity of about 62.35 g/g. However, the dried (to a constant mass) BC-films later exhibited a low water absorption capacity (3.82 g/g). It was found that decellularized BC samples had 2.4 times larger Young's modulus and 2.2 times greater tensile strength as compared to dehydrated native BC films. We presume that this was caused by molecular compaction of the BC structure.

Hepatocellular carcinoma (HCC) is the most common hepatobiliary malignancy with limited therapeutic options. On the other hand, melatonin is an indoleamine that modulates a variety of potential therapeutic effects. In addition to its important role in the regulation of sleep-wake rhythms, several previous studies linked the biologic effects of melatonin to various substantial endocrine, neural, immune and antioxidant functions, among others. Furthermore, the effects of melatonin could be influenced through receptor dependent and receptor independent manner. Among the other numerous physiological and therapeutic effects of melatonin, controlling the survival and differentiation of mesenchymal stem cells (MSCs) has been recently discussed. Given its controversial interaction, several previous reports revealed the therapeutic potential of MSCs in controlling the hepatocellular carcinoma (HCC). Taken together, the intention of the present review is to highlight the effects of melatonin and mesenchymal stem cells as a key for functional integrity for liver cancer treatment. We hope to provide solid piece of information that may be helpful in designing novel drug targets to control HCC.

While droplet-based microfluidics is a powerful technique with transformative applications, most devices are passively operated and thus have limited real-time control over droplet contents. In this report, an automated droplet-based microfluidic device with pneumatic pumps and salt water electrodes was developed to generate and coalesce up to six aqueous-in-oil droplets (2.77 nL each). Custom control software combined six droplets drawn from any of four inlet reservoirs. Using our μChopper method for lock-in fluorescence detection, we first accomplished continuous linear calibration and quantified an unknown sample. Analyte-independent signal drifts and even an abrupt decrease in excitation light intensity were corrected in real-time. The system was then validated with homogeneous insulin immunoassays that showed a nonlinear response. On-chip droplet merging with antibody-oligonucleotide (Ab-oligo) probes, insulin standards, and buffer permitted the real-time calibration and correction of large signal drifts. Full calibrations (LOD_conc = 2 ng mL^-1 = 300 pM; LOD_amt = 5 amol) required <1 min with merely 13.85 nL of Ab-oligo reagents, giving cost-savings 160-fold over the standard well-plate format while also automating the workflow. This proof-of-concept device-effectively a microfluidic digital-to-analog converter-is readily scalable to more droplets, and it is well-suited for the real-time automation of bioassays that call for expensive reagents.

A 51-year-old woman was admitted to the hospital with abdominal pain, jaundice, and transaminitis. The patient's laboratory results showed elevated liver enzymes, high antinuclear antibodies (ANA) titer, positive anti-smooth muscle antibody, and hypergammaglobulinemia. Given risk factors for HIV infection, an ADVIA Centaur® HIV Antigen/Antibody Combo assay was performed showing a reactive sample with a follow up HIV-1 nucleic acid test (NAT) proving to be negative. Following confirmation of autoimmune hepatitis type I via a liver biopsy, steroids were initiated and significant clinical improvement of symptoms as well as resolution in transaminitis were noted. Autoimmunity is the most likely causative factor in inducing a false positive reactive screening assay. It is important to recognize that cross-reactivity with autoimmune conditions and HIV specific proteins is a potential concern for false reactive samples.

All RNA viruses encode the RNA-dependent RNA polymerase (RdRp) which replicates and transcribes viral RNA. This essential viral enzyme does not exist in mammalian cells, thus presents a main target for the development of antiviral drugs with potential pan-antiviral activity. In this study, we take advantage of the structurally equivalent site in the dengue virus (DENV) RdRp, the N-pocket, and in the human norovirus (hNV) RdRp, the B-site, and performed a parallel structure-based virtual screening to discover compounds that can inhibit the RdRps of both hNV and DENV. We successfully identified a small molecule called Entrectinib (RAI-13) as a potent inhibitor of both hNV and DENV infection. Specifically, RAI-13 binds directly to hNV and DENV RdRps, effectively inhibits the polymerase activity in the <i>in vitro</i> biochemical assays, and exhibits does-responsive inhibition of murine norovirus (MNV) and DENV2 infection with IC50 values of 2.01 and 2.43 μM, respectively. Most promisingly, RAI-13 inhibits hepatitis C virus (HCV) infection by 95% at the 2 μM concentration. We have therefore discovered a small molecule compound that targets an allosteric site that is shared by different viral RdRps and strongly inhibits multiple pathogenic RNA viruses, thus holding the potential of being developed into a broad-spectrum antiviral drug.

Seed treatments with antagonistic bacteria could reduce the severity of crown and root rot diseases in wheat crops. The objective of this study was to evaluate the potential antagonistic activity of a bacterial consortium of three Chilean strains of <i>Pseudomonas protegens</i> against the wheat crown and root rot pathogens <i>Gaeumannomyces graminis</i> var. <i>tritici</i>, <i>Rhizoctonia cerealis</i>, and <i>Fusarium culmorum</i>. Two field experiments were carried out on artificially infested soil during two consecutive seasons (2016-2017 and 2017-2018) in an Andisol soil of southern Chile. Control treatments (not inoculated with fungi) were also included. Each treatment included a seed treatment of spring wheat cv. Pantera-INIA with and without the bacterial consortium. Both phytosanitary damage (incidence and severity) and agronomic components were evaluated. Bacterial populations with the <i>phlD</i>+ gene in the wheat plant rhizosphere during anthesis state (Z6) were also quantified. In both seasons, infection severity decreased by an average of 16.8% in seeds treated with <i>P. protegens</i> consortium, while yield components such as spikes m^-1 and number of grains per spike increased. The use of antagonistic bacteria resulted in a total yield increase only during the first experimental season (P < 0.05). In general, accumulated rainfall influenced the antagonistic effect of the consortium of <i>P. protegens</i> strains, accounting for the differences observed between the two seasons. The results suggest that this <i>P. protegens</i> consortium applied on seeds can promote plant growth and protect wheat crops against crown and root rot pathogens in Southern Chile under field conditions.

The fine arrangement of neuronal connectivity during development involves the coordinated action of guidance cues and their receptors. In adolescence, the dopamine circuitry is still developing, with mesolimbic dopamine axons undergoing target-recognition events in the nucleus accumbens (NAcc), while mesocortical projections continue to grow toward the prefrontal cortex (PFC) until adulthood. This segregation of mesolimbic versus mesocortical dopamine pathways is mediated by the guidance cue receptor DCC, which signals dopamine axons intended to innervate the NAcc to recognize this region as their final target. Whether DCC-dependent mesolimbic dopamine axon targeting in adolescence requires the action of its ligand, Netrin-1, is unknown. Here we combined shRNA strategies, quantitative analysis of pre- and post-synaptic markers of neuronal connectivity, and pharmacological manipulations to address this question. Similar to DCC levels in the ventral tegmental area, Netrin-1 expression in the NAcc is dynamic across postnatal life, transitioning from high to low expression across adolescence. Silencing Netrin-1 in the NAcc in adolescence results in an increase in the expanse of the dopamine input to the PFC in adulthood, with a corresponding increase in the number of presynaptic dopamine sites. This manipulation also results in altered dendritic spine density and morphology of medium spiny neurons in the NAcc in adulthood and in reduced sensitivity to the behavioral activating effects of the stimulant drug of abuse, amphetamine. These cellular and behavioral effects mirror those induced by <i>Dcc</i> haploinsufficiency within dopamine neurons in adolescence. Dopamine targeting in adolescence requires the complementary interaction between DCC receptors in mesolimbic dopamine axons and Netrin-1 in the NAcc. Factors regulating either DCC or Netrin-1 in adolescence can disrupt mesocorticolimbic dopamine development, rendering vulnerability or protection to phenotypes associated with psychiatric disorders.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that predominantly affects dopaminergic (DA) neurons of the substantia nigra. Current treatment options for PD are symptomatic and typically involve the replacement of DA neurotransmission by DA drugs, which relieve the patients of some of their motor symptoms. However, by the time of diagnosis, patients have already lost about 70% of their substantia nigra DA neurons and these drugs offer only temporary relief. Therefore, cell replacement therapy has garnered much interest as a potential treatment option for PD. Early studies using human fetal tissue for transplantation in PD patients provided proof of principle for cell replacement therapy, but they also highlighted the ethical and practical difficulties associated with using human fetal tissue as a cell source. In recent years, advancements in stem cell research have made human pluripotent stem cells (hPSCs) an attractive source of material for cell replacement therapy. Studies on how DA neurons are specified and differentiated in the developing mouse midbrain have allowed us to recapitulate many of the positional and temporal cues needed to generate DA neurons <i>in vitro</i>. However, little is known about the developmental programs that govern human DA neuron development. With the advent of single-cell RNA sequencing (scRNA-seq) and bioinformatics, it has become possible to analyze precious human samples with unprecedented detail and extract valuable high-quality information from large data sets. This technology has allowed the systematic classification of cell types present in the human developing midbrain along with their gene expression patterns. By studying human development in such an unbiased manner, we can begin to elucidate human DA neuron development and determine how much it differs from our knowledge of the rodent brain. Importantly, this molecular description of the function of human cells has become and will increasingly be a reference to define, evaluate, and engineer cell types for PD cell replacement therapy and disease modeling.

<title>Abstract</title> <p><bold>Background</bold> The precise epigenetic pathways and mechanisms involved in determining the fate of neural progenitor stem cells (NPCs) into differentiated neural lineages including oligodendrocytes (OLs) is an unresolved issue. MiRNAs are one of the key differentiating factors whose aberrant expression has been shown to disturb proper development. In the present study the leading role of miR-19 in NPCs differentiation to OLs was investigated.<bold>Methods</bold> MiR-19 was overexpressed in hiPSC derived NPCs and for confirming differentiation to the desired cell line, i.e. OL, the expression of cell line-specific genes was evaluated by Real-Time PCR. Oligodendrocyte progenitor cells (OPCs) precursor in the differentiated cells was studied by Flow cytometry. Luciferase activity was also measured to confirm the interaction of miR-19 with the 3’UTR of mRNA of the putative genes. <bold>Results</bold> Our results indicate that the upregulation of miR-19 is a contributing factor in determining NPCs' fate and the timing of progenitor division arrest. MiR-19 can cause a significant expression of OL markers. Besides, miR-19 repressed proneuronal differentiation factors including NeuroD1, NeuroD4, and neurogenin. Moreover, the higher expression of miR-19 could downregulate SOX4, SOX5, and SOX6 genes, which are involved in inhibiting OL differentiation and maturation. <bold>Conclusions</bold> Our finding demonstrated the functional link of miR-19 to regulatory networks of NPCs differentiation, as well as introduced a new strategy for modulating OL fate commitment and development of novel cell therapies.</p>

Aldosterone is produced by the mammalian adrenal cortex to modulate blood pressure and fluid balance; however, excessive, prolonged aldosterone promotes fibrosis and kidney failure. How aldosterone triggers disease may involve actions independent of its canonical mineralocorticoid receptor. Here, we present a <i>Drosophila</i> model of renal pathology caused by excess extracellular matrix formation, stimulated by exogenous aldosterone and by insect ecdysone. Chronic administration of aldosterone or ecdysone induces expression and accumulation of collagen-like Pericardin in adult nephrocytes - podocyte-like cells that filter circulating hemolymph. Excess Pericardin deposition disrupts nephrocyte (glomerular) filtration and causes proteinuria in <i>Drosophila</i>, hallmarks of mammalian kidney failure. Steroid-induced Pericardin production arises from cardiomyocytes associated with nephrocytes, potentially reflecting an analogous role of mammalian myofibroblasts in fibrotic disease. Remarkably, the canonical ecdysteroid nuclear hormone receptor, Ecdysone receptor (EcR), is not required for aldosterone or ecdysone to stimulate Pericardin production or associated renal pathology. Instead, these hormones require a cardiomyocyte-associated G-protein-coupled receptor, Dopamine-EcR (DopEcR), a membrane-associated receptor previously characterized in the fly brain to affect behavior. DopEcR in the brain is known to affect behavior through interactions with the <i>Drosophila</i> Epidermal growth factor receptor (Egfr), referred to as dEGFR. Here, we find that the steroids ecdysone and aldosterone require dEGFR in cardiomyocytes to induce fibrosis of the cardiac-renal system. In addition, endogenous ecdysone that becomes elevated with age is found to foster age-associated fibrosis, and to require both cardiomyocyte DopEcR and dEGFR. This <i>Drosophila</i> renal disease model reveals a novel signaling pathway through which steroids may modulate mammalian fibrosis through potential orthologs of DopEcR.

Tilimycin is an enterotoxin produced by the opportunistic pathogen <i>Klebsiella oxytoca</i> that causes antibiotic-associated hemorrhagic colitis (AAHC). This pyrrolobenzodiazepine (PBD) natural product is synthesized by a bimodular nonribosomal peptide synthetase (NRPS) pathway composed of three proteins: NpsA, ThdA, and NpsB. We describe the functional and structural characterization of the fully reconstituted NRPS system and report the steady-state kinetic analysis of all natural substrates and cofactors as well as the structural characterization of both NpsA and ThdA. The mechanism of action of tilimycin was confirmed using DNA adductomics techniques through the detection of putative N-2 guanine alkylation after tilimycin exposure to eukaryotic cells, providing the first structural characterization of a PBD-DNA adduct formed in cells. Finally, we report the rational design of small-molecule inhibitors that block tilimycin biosynthesis in whole cell <i>K. oxytoca</i> (IC₅₀ = 29 ± 4 μM) through the inhibition of NpsA (<i>K</i>_D = 29 ± 4 nM).

<h4>Lessons learned</h4>Monotherapy with prexasertib demonstrated modest activity in BRCA wild-type, recurrent triple-negative breast cancer, highlighting the unmet need for combination treatment strategies. Neutropenia, anemia, and thrombocytopenia are common with the use of prexasertib but are manageable with supportive care measures. Prophylactic use of granulocyte colony stimulating factor should be considered to avoid dose reductions or treatment delays. Pharmacodynamic studies showed prexasertib treatment induced DNA damage in peripheral immune cells.<h4>Background</h4>Cell cycle checkpoint kinase 1 (CHK1) is a major G2/M cell cycle regulator in tumors with p53 dysfunction, such as triple-negative breast cancer (TNBC). We hypothesized the second-generation CHK1 inhibitor, prexasertib, would yield clinical activity in sporadic TNBC.<h4>Methods</h4>This single arm, phase II trial evaluated prexasertib at 105 mg/m² IV every 2 weeks in patients with metastatic/recurrent TNBC. The primary endpoint was overall response rate (ORR).<h4>Results</h4>All nine patients enrolled were germline BRCA wild-type (BRCAwt) and had at least one prior treatment. One partial response (PR) was observed (ORR of 11.1%). Four patients experienced stable disease. The median progression-free survival (PFS) was 86 days (range 17 to 159 days). Grade 3/4 treatment-related adverse events included afebrile neutropenia (n = 8; 88.9%), anemia (n = 3; 33.3%), and thrombocytopenia (n = 1; 11.1%). Pharmacodynamic studies showed prexasertib treatment induced DNA damage in peripheral immune cells and demonstrated a decrease in activated/reinvigorated CD8 T cells; however, the one patient with a PR showed evidence of T-cell recovery.<h4>Conclusion</h4>Prexasertib monotherapy had modest clinical efficacy in BRCAwt TNBC. Further studies of prexasertib in combination with other agents are needed.

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a 'PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

Bovine leukemia virus (BLV) is a δ-retrovirus responsible for Enzootic Bovine Leukosis (EBL), a lymphoproliferative disease that affects cattle. The virus causes immune system deregulation, favoring the development of secondary infections. In that context, mastitis incidence is believed to be increased in BLV infected cattle. The aim of this study was to analyze the transcriptome profile of a BLV infected mammary epithelial cell line (MAC-T). Our results show that BLV infected MAC-T cells have an altered expression of IFN I signal pathway and genes involved in defense response to virus, as well as a collagen catabolic process and some protooncogenes and tumor suppressor genes. Our results provide evidence to better understand the effect of BLV on bovine mammary epithelial cell's immune response.

<h4>Background</h4>We previously reported an inverse relationship between B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and Raf kinase inhibitory protein (RKIP), which is associated with the prognosis of gastric cancer (GC). In this study, we further explored the microRNA (miRNA) regulatory mechanism between Bmi-1 and RKIP.<h4>Methods</h4>Microarray analysis was first carried out to identify miRNA profiles that were differentially expressed in cells overexpressing Bmi-1. Then, miRNAs that could regulate RKIP were identified. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to measure the expression of Bmi-1, miR-155, miR-27a and RKIP. RKIP was confirmed as a target of miR-27a and miR-155 through luciferase reporter assays, qRT-PCR and Western blotting. The effects of the Bmi-1/miR-27a/RKIP and Bmi-1/miR-155/RKIP axes on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both in vitro and in vivo.<h4>Results</h4>The downregulation of RKIP by Bmi-1 occurred at the protein but not mRNA level. This indicates probable posttranscriptional regulation. miRNA expression profiles of cells with ectopic expression of Bmi-1 were analyzed and compared to those of control cells by microarray analysis. A total of 51 upregulated and 72 downregulated miRNAs were identified. Based on publicly available algorithms, miR-27a and miR-155 were predicted, selected and demonstrated to target RKIP. Bmi-1, miR-27a and miR-155 are elevated in human GC and associated with poor prognosis of GC, while RKIP is expressed at lower levels in GC and correlated with good prognosis. Then, in vitro tests shown that in addition to regulating RKIP expression via miR-27a and miR-155, Bmi-1 was also able to regulate the migration, invasion, proliferation, colony-formation ability and chemosensitivity of GC cells through the same pathway. Finally, the in vivo test showed similar results, whereby the knockdown of the Bmi-1 gene led to the inhibition of tumor growth, metastasis and chemoresistance through miR-27a and miR-155.<h4>Conclusions</h4>Bmi-1 was proven to induce the expression of miR-27a and miR-155 and thus promote tumor metastasis and chemoresistance by targeting RKIP in GC. Overall, miR-27a and miR-155 might be promising targets for the screening, diagnosis, prognosis, treatment and disease monitoring of GC.

<h4>Background</h4>Exome and genome sequencing have been demonstrated to increase diagnostic yield in paediatric populations, improving treatment options and providing risk information for relatives. There are limited studies examining the clinical utility of these tests in adults, who currently have limited access to this technology.<h4>Methods</h4>Patients from adult and cancer genetics clinics across Toronto, Ontario, Canada were recruited into a prospective cohort study evaluating the diagnostic utility of exome and genome sequencing in adults. Eligible patients were ≥18 years of age and suspected of having a hereditary disorder but had received previous uninformative genetic test results. In total, we examined the diagnostic utility of exome and genome sequencing in 47 probands and 34 of their relatives who consented to participate and underwent exome or genome sequencing.<h4>Results</h4>Overall, 17% (8/47) of probands had a pathogenic or likely pathogenic variant identified in a gene associated with their primary indication for testing. The diagnostic yield for patients with a cancer history was similar to the yield for patients with a non-cancer history (4/18 (22%) vs 4/29 (14%)). An additional 24 probands (51%) had an inconclusive result. Secondary findings were identified in 10 patients (21%); three had medically actionable results.<h4>Conclusions</h4>This study lends evidence to the diagnostic utility of exome or genome sequencing in an undiagnosed adult population. The significant increase in diagnostic yield warrants the use of this technology. The identification and communication of secondary findings may provide added value when using this testing modality as a first-line test.

Huntington disease (HD) is caused by an expansion mutation of the N-terminal polyglutamine of huntingtin (mHTT). mHTT is ubiquitously present, but it induces noticeable damage to the brain's striatum, thereby affecting motor, psychiatric, and cognitive functions. The striatal damage and progression of HD are associated with the inflammatory response; however, the underlying molecular mechanisms remain unclear. Here, we report that cGMP-AMP synthase (cGAS), a DNA sensor, is a critical regulator of inflammatory and autophagy responses in HD. Ribosome profiling revealed that the <i>cGAS</i> mRNA has high ribosome occupancy at exon 1 and codon-specific pauses at positions 171 (CCG) and 172 (CGT) in HD striatal cells. Moreover, the protein levels and activity of cGAS (based on the phosphorylated STING and phosphorylated TBK1 levels), and the expression and ribosome occupancy of cGAS-dependent inflammatory genes (<i>Ccl5</i> and <i>Cxcl10</i>) are increased in HD striatum. Depletion of cGAS diminishes cGAS activity and decreases the expression of inflammatory genes while suppressing the up-regulation of autophagy in HD cells. In contrast, reinstating cGAS in cGAS-depleted HD cells activates cGAS activity and promotes inflammatory and autophagy responses. Ribosome profiling also revealed that <i>LC3A</i> and <i>LC3B</i>, the two major autophagy initiators, show altered ribosome occupancy in HD cells. We also detected the presence of numerous micronuclei, which are known to induce cGAS, in the cytoplasm of neurons derived from human HD embryonic stem cells. Collectively, our results indicate that cGAS is up-regulated in HD and mediates inflammatory and autophagy responses. Thus, targeting the cGAS pathway may offer therapeutic benefits in HD.

Breast cancer is the most common type of cancer worldwide and one of the major causes of cancer death in women. Epidemiological studies have established a link between night-shift work and increased cancer risk, suggesting that circadian disruption may play a role in carcinogenesis. Here, we aim to shed light on the effect of chronic jetlag (JL) on mammary tumour development. To do this, we use a mouse model of spontaneous mammary tumourigenesis and subject it to chronic circadian disruption. We observe that circadian disruption significantly increases cancer-cell dissemination and lung metastasis. It also enhances the stemness and tumour-initiating potential of tumour cells and creates an immunosuppressive shift in the tumour microenvironment. Finally, our results suggest that the use of a CXCR2 inhibitor could correct the effect of JL on cancer-cell dissemination and metastasis. Altogether, our data provide a conceptual framework to better understand and manage the effects of chronic circadian disruption on breast cancer progression.

Pan-cancer genomic analyses based on the magnitude of pathway activity are currently lacking. Focusing on the cell cycle, we examined the DNA mutations and chromosome arm-level aneuploidy within tumours with low, intermediate and high cell-cycle activity in 9515 pan-cancer patients with 32 different tumour types. Boxplots showed that cell-cycle activity varied broadly across and within all cancers. TP53 and PIK3CA mutations were common in all cell cycle score (CCS) tertiles but with increasing frequency as cell-cycle activity levels increased (P < 0.001). Mutations in BRAF and gains in 16p were less frequent in CCS High tumours (P < 0.001). In Kaplan-Meier analysis, patients whose tumours were CCS Low had a longer Progression Free Interval (PFI) relative to Intermediate or High (P < 0.001) and this significance remained in multivariable analysis (CCS Intermediate: HR = 1.37; 95% CI 1.17-1.60, CCS High: 1.54; 1.29-1.84, CCS Low = Ref). These results demonstrate that whilst similar DNA alterations can be found at all cell-cycle activity levels, some notable exceptions exist. Moreover, independent prognostic information can be derived on a pan-cancer level from a simple measure of cell-cycle activity.

Expression quantitative trait locus (eQTL) analyses have enabled us to predict the function of disease susceptibility SNPs. However, eQTL for the effector memory T cells (TEM) located in the lamina propria mononuclear cells (LPMCs), which play an important role in Crohn's disease (CD), are not yet available. Thus, we conducted RNA sequencing and eQTL analyses of TEM cells located in the LPMCs from IBD patients (n = 20). Genome-wide association study (GWAS) was performed using genotyping data of 713 Japanese CD patients and 2,063 controls. We compared the results of GWAS and eQTL of TEM, and also performed a transcriptome-wide association study using eQTL from Genotype Tissue Expression project. By eQTL analyses of TEM, correlations of possible candidates were confirmed in 22,632 pairs and 2,463 genes. Among these candidates, 19 SNPs which showed significant correlation with tenascin-XA (TNXA) expression were significantly associated with CD in GWAS. By TWAS, TNFSF15 (FDR = 1.35e-13) in whole blood, ERV3-1 (FDR = 2.18e-2) in lymphocytes, and ZNF713 (FDR = 3.04e-2) in the sigmoid colon was significantly associated with CD. By conducting integration analyses using GWAS and eQTL data, we confirmed multiple gene transcripts are involved in the development of CD.

Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra¹. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits². Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson's disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.

Carbonic anhydrase 8 (CA8), an isozyme of α-carbonic anhydrases, lacks the ability to catalyze the reversible hydration of CO₂ to bicarbonate and proton. Previous studies have shown that single point mutations of CA8, CA8-S100P, and CA8-G162R, are associated with novel syndromes including congenital ataxia and mild cognitive impairment. Our previous results demonstrated that overexpression of wild type (WT) CA8 promoted cell proliferation, neurite outgrowth, anti-apoptosis, invasion and migration abilities in neuronal cells. In this study, we examined the expressions and functions of CA8-S100P and CA8-G162R in neuroblastoma cells lines, compared with those of WT CA8. Our results show that the protein expressions of mutant CA8-S100P and CA8-G162R were significantly decreased in Neuro-2a and SK-N-SH cells. Interestingly, CA8-S100P demonstrated a significant increase in cell proliferation in both Neuro-2a and SK-N-SH cells. However, both CA8 mutations showed significantly decreased effects on cell protection and migration in SK-N-SH cells. Surprisingly, a significant increase of invasive ability was observed in SK-N-SH cells with overexpression of CA8-S100P as compared with those with overexpression of WT CA8 under retinoic acid (RA) treatment. In addition, we found that Neuro-2a cells with overexpression of CA8-S100P and CA8-G162R showed significantly increased neurite outgrowth. Taken together, our data suggest that the expressions of CA8-S100P and CA8-G162R in neuronal cells alter cell morphology, proliferation, mobility and viability; indicating that the homozygous point mutations of CA8 lead to not only the loss of WT CA8 function, but also the gain of novel functions leading to neuromuscular dysfunction.

COVID-19 pandemia is a major health emergency causing hundreds of deaths worldwide. The high reported morbidity has been related to hypoxia and inflammation leading to endothelial dysfunction and aberrant coagulation in small and large vessels. This review addresses some of the pathways leading to endothelial derangement, such as complement, HIF-1α, and ABL tyrosine kinases. This review also highlights potential targets for prevention and therapy of COVID-19-related organ damage and discusses the role of marketed drugs, such as eculizumab and imatinib, as suitable candidates for clinical trials.

The identification of rare disease-causing variants in humans by large-scale next-generation sequencing (NGS) studies has also provided us with new insights into the pathophysiological role of de novo missense variants in the CACNA1D gene that encodes the pore-forming α1-subunit of voltage-gated Cav1.3 L-type Ca²⁺ channels. These CACNA1D variants have been identified somatically in aldosterone-producing adenomas as well as germline in patients with neurodevelopmental and in some cases endocrine symptoms. In vitro studies in heterologous expression systems have revealed typical gating changes that indicate enhanced Ca²⁺ influx through Cav1.3 channels as the underlying disease-causing mechanism. Here we summarize the clinical findings of 12 well-characterized individuals with a total of 9 high-risk pathogenic CACNA1D variants. Moreover, we propose how information from somatic mutations in aldosterone-producing adenomas could be used to predict the potential pathogenicity of novel germline variants. Since these pathogenic de novo variants can cause a channel-gain-of function, we also discuss the use of L-type Ca²⁺ channel blockers as a potential therapeutic option.

Common sequence variations in the VRK2 gene contribute to genetic risk for various psychiatric diseases including schizophrenia and major depressive disorder. Despite the clear importance of studying the regulation and function of VRK2 for understanding the causes of these diseases, the organisation and expression of the gene remain poorly characterised. Using reverse-transcriptase-PCR, we have amplifed exons of Vrk2 mRNA from regions of mouse brain, and from different cell classes comprising neurones, astrocytes and microglial cells. We find that Vrk2 mRNA is expressed in all cell types, and that the splicing of the mouse Vrk2 gene is much more complex than previously appreciated. In addition to the predicted alternative splicing (absence/presence) of the penultimate 3 prime exon, we also detected a variety of 5 prime structures, including two novel exons spanning the first characterised exon (exon 1), which we term exons 1a and 1b. While expressed in neurones and astrocytes, exon 1b was not expressed in microglial cells. Expression of transcripts containing exon 1a in microglia was increased by immune stimulation. An additional truncated transcript lacking 7 central exons was also identified. As with the human gene, the results confirm complex patterns of alternative splicing which are likely to be relevant for understanding the physiological and pathological function of the gene in the CNS.

Heparin resistance is an uncommon phenomenon defined as the need for high-dose unfractionated heparin (UFH) of more than 35,000 IU/day to achieve the target activated partial-thromboplastin time ratio or the failure to achieve the desired activated clotting time after a full UFH dose. This rare phenomenon is being more commonly observed in Covid-19 patients in a hypercoagulable state. We describe a Covid-19 patient confirmed by reverse-transcriptase polymerase chain reaction assay, with acute limb ischemia, who developed heparin resistance. The patient was managed by the departments of vascular surgery, anesthesia and intensive care, and the Coagulation Service and Thrombosis Research from San Raffaele Scientific Institute, Milan, Italy.

<h4>Background</h4>A subset of patients have no risk factors for the development of hepatocellular carcinoma (HCC). We evaluated differences in clinical variables between patients with and without risk factors who underwent surgical resection.<h4>Methods</h4>A prospectively maintained database was queried for patients who underwent R0/R1 resection of HCC between 1992 and 2016. Risk factors included HCV, HBV, hemochromatosis, alcoholic liver disease, or cirrhosis, stage 2 or 3 fibrosis or severe (>66%) steatosis of the non-neoplastic liver. Variables were compared between patients with and without risk factors.<h4>Results</h4>There were 416 patients who underwent resection; 276 (66%) had known risk factors while 140 (34%) did not. Patients without risk factors were more likely to be older, female and have hyperlipidemia or coronary artery disease (p < 0.004). These patients had larger tumors and were more likely to undergo major hepatectomy (p < 0.001). There was no difference in OS (5-year, 56% vs 47%, p = 0.335), RFS (27% vs 24%, p = 0.398), or the rates of intrahepatic (HR:1.16 [95%CI:0.95-1.57], p = 0.344) and extrahepatic recurrences (HR:0.72 [95%CI:0.4-1.3], p = 0.261) between groups.<h4>Conclusion</h4>Patients without risk factors for HCC presented with larger tumors yet had similar outcomes, suggesting these tumors may represent a different disease process, and underlying liver dysfunction can influence overall outcome.

Throughout life, organisms are exposed to various exogenous and endogenous factors that cause DNA damages and somatic mutations provoking genomic instability. At a young age, compensatory mechanisms of genome protection are activated to prevent phenotypic and functional changes. However, the increasing stress and age-related deterioration in the functioning of these mechanisms result in damage accumulation, overcoming the functional threshold. This leads to aging and the development of age-related diseases. There are several ways to counteract these changes: 1) prevention of DNA damage through stimulation of antioxidant and detoxification systems, as well as transition metal chelation; 2) regulation of DNA methylation, chromatin structure, non-coding RNA activity and prevention of nuclear architecture alterations; 3) improving DNA damage response and repair; 4) selective removal of damaged non-functional and senescent cells. In the article, we have reviewed data about the effects of various trace elements, vitamins, polyphenols, terpenes, and other phytochemicals, as well as a number of synthetic pharmacological substances in these ways. Most of the compounds demonstrate the geroprotective potential and increase the lifespan in model organisms. However, their genome-protecting effects are non-selective and often are conditioned by hormesis. Consequently, the development of selective drugs targeting genome protection is an advanced direction.

Abdominal artery aneurysm (AAA) refers to abdominal aortic dilatation of 3 cm or greater. AAA is frequently underdiagnosed due to often asymptomatic character of the disease, leading to elevated mortality due to aneurysm rupture. MiRNA constitute a pool of small RNAs controlling gene expression and is involved in many pathologic conditions in human. Targeted panel detecting altered expression of miRNA and genes involved in AAA would improve early diagnosis of this disease. In the presented study, we selected and analyzed miRNA and gene expression signatures in AAA patients. Next, generation sequencing was applied to obtain miRNA and gene-wide expression profiles from peripheral blood mononuclear cells in individuals with AAA and healthy controls. Differential expression analysis was performed using DESeq2 and uninformative variable elimination by partial least squares (UVE-PLS) methods. A total of 31 miRNAs and 51 genes were selected as the most promising biomarkers of AAA. Receiver operating characteristics (ROC) analysis showed good diagnostic ability of proposed biomarkers. Genes regulated by selected miRNAs were determined in silico and associated with functional terms closely related to cardiovascular and neurological diseases. Proposed biomarkers may be used for new diagnostic and therapeutic approaches in management of AAA. The findings will also contribute to the pool of knowledge about miRNA-dependent regulatory mechanisms involved in pathology of that disease.

Black pepper (<i>Piper nigrum</i> L.) has been employed in medicine (epilepsy, headaches, and diabetes), where its effects are mainly attributed to a nitrogen alkaloid called piperidine (1-(1-[1,3-benzodioxol-5-yl]-1-oxo-2,4 pentenyl) piperidine). Piperine co-administered with vitamins and minerals has improved its absorption. Therefore, this study aimed to describe the impact of the joint administration of iron (Fe) plus black pepper in physically active healthy individuals. Fe is a micronutrient that aids athletic performance by influencing the physiological functions involved in endurance sports by improving the transport, storage, and utilization of oxygen. Consequently, athletes have risk factors for Fe depletion, Fe deficiency, and eventually, anemia, mainly from mechanical hemolysis, gastrointestinal disturbances, and loss of Fe through excessive sweating. Declines in Fe stores have been reported to negatively alter physical capacities such as aerobic capacity, strength, and skeletal muscle recovery in elite athletes. Thus, there is a need to maintain Fe storage, even if Fe intake meets the recommended daily allowance (RDA), and Fe supplementation may be justified in physically active individuals, in states of Fe deficiency, with or without anemia. Females, in particular, should monitor their Fe hematological profile. The recommended oral Fe supplements are ferrous or ferric salts, sulfate, fumarate, and gluconate. These preparations constitute the first line of treatment; however, the high doses administered have gastrointestinal side effects that reduce tolerance and adherence to treatment. Thus, a strategy to counteract these adverse effects is to improve the bioavailability of Fe. Therefore, piperine may benefit the absorption of Fe through its bioavailability enhancement properties. Three research studies of Fe associated with black pepper have reported improvements in parameters related to the metabolism of Fe, without adverse effects. Although more research is needed, this could represent an advance in oral Fe supplementation for physically active individuals.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs). Patients with glaucoma generally experience elevations in intraocular pressure (IOP), followed by RGC death, peripheral vision loss and eventually blindness. However, despite the substantial economic and health-related impact of glaucoma-related morbidity worldwide, the surgical and pharmacological management of glaucoma is still limited to maintaining IOP within a normal range. This is in large part because the underlying molecular and biophysical mechanisms by which glaucomatous changes occur are still unclear. In the present review article, we describe current tissue-engineered models of the intraocular space that aim to advance the state of glaucoma research. Specifically, we critically evaluate and compare both 2D and 3D-culture models of the trabecular meshwork and nerve fiber layer, both of which are key players in glaucoma pathophysiology. Finally, we point out the need for novel organ-on-a-chip models of glaucoma that functionally integrate currently available 3D models of the retina and the trabecular outflow pathway.

G protein coupled receptors (GPCRs) are the main mediators of signal transduction in the central nervous system. Therefore, it is not surprising that many GPCRs have long been investigated for their role in the development of anxiety and mood disorders, as well as in the mechanism of action of antidepressant therapies. Importantly, the endogenous ligands for a large group of GPCRs have not yet been identified and are therefore known as orphan GPCRs (oGPCRs). Nonetheless, growing evidence from animal studies, together with genome wide association studies (GWAS) and post-mortem transcriptomic analysis in patients, pointed at many oGPCRs as potential pharmacological targets. Among these discoveries, we summarize in this review how emotional behaviors are modulated by the following oGPCRs: ADGRB2 (BAI2), ADGRG1 (GPR56), GPR3, GPR26, GPR37, GPR50, GPR52, GPR61, GPR62, GPR88, GPR135, GPR158, and GPRC5B.

Colorectal carcinogenesis (CRC) is caused by the gradual long-term accumulation of both genetic and epigenetic changes. Recently, epigenetic alterations have been included in the classification of the CRC molecular subtype, and this points out their prognostic impact. As epigenetic modifications are reversible, they may represent relevant therapeutic targets. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), regulates gene expression. For many years, the deregulation of DNA methylation has been considered to play a substantial part in CRC etiology and evolution. Despite considerable advances in CRC treatment, patient therapy response persists as limited, and their profit from systemic therapies are often hampered by the introduction of chemoresistance. In addition, inter-individual changes in therapy response in CRC patients can arise from their specific (epi)genetic compositions. In this review article, we summarize the options of CRC treatment based on DNA methylation status for their predictive value. This review also includes the therapy outcomes based on the patient's methylation status in CRC patients. In addition, the current challenge of research is to develop therapeutic inhibitors of DNMT. Based on the essential role of DNA methylation in CRC development, the application of DNMT inhibitors was recently proposed for the treatment of CRC patients, especially in patients with DNA hypermethylation.

Soft tissue sarcomas (STS) are rare, malignant tumours with a generally poor prognosis. Our aim was to explore the potential of cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) analysis to track non-metastatic STS patients undergoing attempted curative treatment. The analysed cohort (<i>n</i> = 29) contained multiple STS subtypes including myxofibrosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, and dedifferentiated liposarcomas amongst others. Perioperative cfDNA levels trended towards being elevated in patients (<i>p</i> = 0.07), although did not correlate with tumour size, grade, recurrence or subtype, suggesting a limited diagnostic or prognostic role. To characterise ctDNA, an amplicon panel covering three genes commonly mutated in STSs was first trialled on serial plasma collected from nine patients throughout follow-up. This approach only identified ctDNA in 2.5% (one in 40) of the analysed samples. Next custom-designed droplet digital PCR assays and Ion AmpliSeq™ panels were developed to track single nucleotide variants identified in patients' STSs by whole exome sequencing (1-6 per patient). These approaches identified ctDNA in 17% of patients. Although ctDNA was identified before radiologically detectable recurrence in two cases, the absence of demonstrable ctDNA in 83% of cases highlights the need for much work before circulating nucleic acids can become a useful means to track STS patients.

At present, studies on macrophage proteins mainly focus on biological stimuli, with less attention paid to the responses of macrophage proteins to physical stimuli, such as electric fields. Here, we exploited the electric field-sensitive hub proteins of macrophages. RAW 264.7 macrophages were treated with a direct current electric field (dcEF) (200 mV/mm) for four hours, followed by RNA-Seq analysis. Differentially expressed genes (DEGs) were obtained, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and protein-protein interaction (PPI) analysis. Eight qPCR-verified DEGs were selected. Subsequently, three-dimensional protein models of DEGs were modeled by Modeller and Rosetta, followed by molecular dynamics simulation for 200 ns with GROMACS. Finally, dcEFs (10, 50, and 500 mV/mm) were used to simulate the molecular dynamics of DEG proteins for 200 ns, followed by trajectory analysis. The dcEF has no obvious effect on RAW 264.7 morphology. A total of 689 DEGs were obtained, and enrichment analysis showed that the steroid biosynthesis pathway was most affected by the dcEF. Moreover, the three-dimensional protein structures of hub proteins were constructed, and trajectory analysis suggested that the dcEF caused an increase in the atomic motion of the protein in a dcEF-intensity-dependent manner. Overall, we provide new clues and a basis for investigating the hub proteins of macrophages in response to electric field stimulation.

In recent years, synthetic calcium phosphate (CaP) ceramics have emerged as an alternative to bone grafts in the treatment of large critical-sized bone defects. To successfully substitute for bone grafts, materials must be osteoinductive, that is, they must induce osteogenic differentiation and subsequent bone formation <i>in vivo</i>. Although a set of osteoinductive CaP ceramics has been developed, the precise biological mechanism by which a material directs cells toward osteogenesis and the role of individual chemical and physical properties in this mechanism remain incompletely understood. Here, we used proteomics to compare serum protein adsorption to two CaP ceramics with different osteoinductive potential, namely an osteoinductive β-tricalcium phosphate (TCP) and a non-osteoinductive hydroxyapatite (HA). Moreover, we analyzed the protein profiles of human mesenchymal stromal cells (hMSCs) cultured on these two ceramics. The serum protein adsorption experiments in the absence of cells highlighted the proteins that are highly abundant in the serum and/or have a high affinity to CaP. The extent of adsorption was suggested to be affected by the available surface area for binding and by the ion exchange dynamics on the surface. Several proteins were uniquely expressed by hMSCs on TCP and HA surfaces. Proteins identified as enriched on TCP were involved in processes related to wound healing, cell proliferation, and the production of extracellular matrix. On the other hand, proteins that were enriched on HA were involved in processes related to protein production, translation, localization, and secretion. In addition, we performed a separate proteomics analysis on TCP, HA, and two biphasic calcium phosphates with known osteoinductive potential and performed a clustering analysis on a combination of a set of proteins found to be enriched on osteoinductive materials with a set of proteins already known to be involved in osteogenesis. This yielded two protein networks potentially involved in the process of osteoinduction - one consisting of collagen fragments and collagen-related enzymes and a second consisting of endopeptidase inhibitors and regulatory proteins. The results of this study show that protein profiling can be a useful tool to help understand the effect of biomaterial properties on the interactions between a biomaterial and a biological system. Such understanding will contribute to the design and development of improved biomaterials for (bone) regenerative therapies.

For hundreds of thousands of years, the human genome has extensively evolved, resulting in genetic variations in almost every gene. Immunological reflections of these genetic variations become clearly visible after an allogeneic stem cell transplantation (allo-SCT) as minor Histocompatibility (H) antigens. Minor H antigens are peptides cleaved from genetically encoded variable protein regions after which they are presented at the cell surface by HLA molecules. After allo-SCT with minor H antigen mismatches between donor and recipient, donor T cells recognize the minor H antigens of the recipient as foreign, evoking strong alloreactive immune responses. Studies in the late eighties have discovered that a subset of minor H antigens are encoded by hematopoietic system-specific genes. After allo-SCT, this subset is strictly expressed on the hematopoietic malignant cells and was therefore the first well-defined highly immunogenic group of tumor-specific antigens. In the last decade, neoantigens derived from genetic mutations in tumors have been identified as another group of immunogenic tumor-specific antigens. Therefore, hematopoietic minor H antigens and neoantigens are therapeutic equivalents. This review will connect our current knowledge about the immune biology and identification of minor H antigens and neoantigens leading to novel conclusions on their prediction.

<h4>Background</h4>A large deletion in Xp22.3 can result in contiguous gene syndromes, including X-linked ichthyosis (XLI) and Kallmann syndrome (KS), presenting with short stature, chondrodysplasia punctata, intellectual disability, and strabismus. XLI and KS are caused by the deletion of <i>STS</i> and <i>ANOS1</i>, respectively.<h4>Method</h4>Two KS patients with XLI were screened to identify possible pathogenic mutations using whole exome sequencing. The clinical characteristics, molecular genetics, treatment outcomes, and genotype-phenotype association for each patient were analyzed.<h4>Results</h4>We identified a novel 3,923 kb deletion within the Xp22.31 region (chrX: 5810838-9733877) containing <i>STS</i>, <i>ANOS1</i>, <i>GPR143</i>, <i>NLGN4X</i>, <i>VCX-A</i>, <i>PUDP</i>, and <i>PNPLA4</i> in patient 1, who presented with KS, XLI, obesity, hyperlipidemia, and strabismus. We identified a novel 5,807 kb deletion within the Xp22.31-p22.33 regions (chrX: 2700083-8507807) containing <i>STS</i>, <i>ANOS1</i>, and other 24 genes in patient 2, who presented with KS, XLI, obesity, and strabismus. No developmental delay, abnormal speech development, or autistic behavior were noticed in either patient.<h4>Conclusion</h4>We identified two novel microdeletions in the X chromosome leading to KS and XLI. These findings contribute to the understanding of the molecular mechanisms that drive contiguous gene syndromes. Our research confirmed that the Kallmann-Ichthyosis phenotype is caused by microdeletions at the chromosome level.

Human mesenchymal stem cells (hMSCs) are a promising candidate in cell therapy as they exhibit multilineage differentiation, homing to the site of injury, and secretion of trophic factors that facilitate tissue healing and/or modulate immune response. As a result, hMSC-derived products have attracted growing interests in preclinical and clinical studies. The development of hMSC culture platforms for large-scale biomanufacturing is necessary to meet the requirements for late-phase clinical trials and future commercialization. Microcarriers in stirred-tank bioreactors have been widely utilized in large-scale expansion of hMSCs for translational applications because of a high surface-to-volume ratio compared to conventional 2D planar culture. However, recent studies have demonstrated that microcarrier-expanded hMSCs differ from dish- or flask-expanded cells in size, morphology, proliferation, viability, surface markers, gene expression, differentiation potential, and secretome profile which may lead to altered therapeutic potency. Therefore, understanding the bioprocessing parameters that influence hMSC therapeutic efficacy is essential for the optimization of microcarrier-based bioreactor system to maximize hMSC quantity without sacrificing quality. In this review, biomanufacturing parameters encountered in planar culture and microcarrier-based bioreactor culture of hMSCs are compared and discussed with specific focus on cell-adhesion surface (e.g., discontinuous surface, underlying curvature, microcarrier stiffness, porosity, surface roughness, coating, and charge) and the dynamic microenvironment in bioreactor culture (e.g., oxygen and nutrients, shear stress, particle collision, and aggregation). The influence of dynamic culture in bioreactors on hMSC properties is also reviewed in order to establish connection between bioprocessing and stem cell function. This review addresses fundamental principles and concepts for future design of biomanufacturing systems for hMSC-based therapy.

Clinical success of chimeric antigen receptor (CAR) T cell immunotherapy requires the engineering of autologous T cells, which limits the broader implementation of CAR cell therapy. The development of allogeneic and universal cell products will significantly broaden their application and reduce costs. Allogeneic natural killer (NK) cells can be used for universal CAR immunotherapy. Here, we develop an alternative approach for the rapid expansion of primary NK and CAR-NK cells with superior expansion capability and <i>in vivo</i> cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, "off-the-shelf" NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.

Transcription factor Yin Yang 1 (YY1) is upregulated in multiple tumors and plays essential roles in tumor proliferation and metastasis. However, the function of YY1 in breast cancer stemness remains unclear. Herein, we found that YY1 expression was negatively correlated with the overall survival and relapse-free survival of breast cancer patients and positively correlated with the expression of stemness markers in breast cancer. Overexpression of YY1 increased the expression of stemness markers, elevated CD44⁺CD24^- cell sub-population, and enhanced the capacity of cell spheroid formation and tumor-initiation. In contrast, YY1 knockdown exhibited the opposite effects. Mechanistically, YY1 decreased microRNA-873-5p (miR-873-5p) level by recruiting histone deacetylase 4 (HDAC4) and HDAC9 to miR-873-5p promoter and thus increasing the deacetylation level of miR-873-5p promoter. Sequentially, YY1 activated the downstream PI3K/AKT and ERK1/2 pathways, which have been confirmed to be suppressed by miR-873-5p in our recent work. Moreover, the suppressed effect of YY1/miR-873-5p axis on the stemness of breast cancer cells was partially dependent on PI3K/AKT and ERK1/2 pathways. Finally, it was found that the YY1/miR-873-5p axis is involved in the chemoresistance of breast cancer cells. Our study defines a novel YY1/miR-873-5p axis responsible for the stemness of breast cancer cells.

An intriguing relationship between menstrual cycle phase at the time of breast cancer surgery and clinical outcomes was first proposed in the late 1980s. Despite a number of clinical studies conducted to address this, as well as meta-analyses and systematic reviews, there remains significant controversy surrounding the effect of menstrual cycle phase at time of surgery on the prognosis of premenopausal breast cancer. While some studies have suggested that surgery performed during the luteal phase results in the most favourable outcome, other studies report the follicular phase is more favourable, and others show no association. Given the conflicting results, there remains insufficient evidence to determine whether there is an optimal time of the month to perform surgery. This issue has dogged breast cancer surgery for decades; knowledge of an optimal time of the month to conduct surgery would be a simple approach to improving patient outcomes. This review explores the potential biological mechanisms through which the hormonal milieu might contribute to differences in prognosis, and why clinical findings are so variable. It is concluded that a significant problem with current clinical research is the lack of insight from mechanistic studies. While there are a number of plausible biological mechanisms that could lead to altered survival, supporting evidence is limited. There are also variable approaches to defining the menstrual cycle phase and hormone receptor status of the tumour and few studies controlled for prognostic factors such as tumour size and stage, or addressed the impact of adjuvant treatments. Elucidation of the specific confounding factors, as well as biological mechanistic pathways that could explain the potential relationship between timing of surgery and survival, will greatly assist in designing robust well-controlled prospective clinical studies to evaluate this paradigm.

<h4>ABSTRACT</h4> Patients with IBD are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the effect of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2 , in IBD. We collected (un)inflamed mucosal biopsies from CD (n=193) and UC (n=158) patients, and 51 matched non-IBD controls for RNA sequencing, differential gene expression and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell (sc) sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-TNF therapy, were analyzed. In inflamed CD ileum, ACE2 was significantly decreased compared to control ileum (p=4.6E-07), whereas colonic ACE2 expression was higher in inflamed colon of CD/UC compared to control (p=8.3E-03; p=1.9E-03). Sc-RNA sequencing confirmed this ACE2 dysregulation, and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2 , while pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids (p=1.7E-02), not in non-IBD controls (p=9.1E-01). Anti-TNF therapy restored colonic ACE2 dysregulation in responders. Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing effects in ileum and colon. HNF4A , an IBD susceptibility gene, is an important upstream regulator of ACE2 in ileum, whereas interferon signaling dominates in colon. Our data support the importance of adequate control of IBD in order to reduce risk of (complicated) COVID-19.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global public health emergency. COVID-19 typically manifests as a respiratory illness but an increasing number of clinical reports describe gastrointestinal (GI) symptoms. This is particularly true in children in whom GI symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. By contrast, fetuses seem to be rarely affected by COVID-19, although the virus has been detected in placentas of affected women. These observations raise the question of whether the virus can infect and replicate within the stomach once ingested. Moreover, it is not yet clear whether active replication of SARS-CoV-2 is possible in the stomach of children or in fetuses at different developmental stages. Here we show the novel derivation of fetal gastric organoids from 8-21 post-conception week (PCW) fetuses, and from pediatric biopsies, to be used as an in vitro model for SARS-CoV-2 gastric infection. Gastric organoids recapitulate human stomach with linear increase of gastric mucin 5AC along developmental stages, and expression of gastric markers pepsinogen, somatostatin, gastrin and chromogranin A. In order to investigate SARS-CoV-2 infection with minimal perturbation and under steady-state conditions, we induced a reversed polarity in the gastric organoids (RP-GOs) in suspension. In this condition of exposed apical polarity, the virus can easily access viral receptor angiotensin-converting enzyme 2 (ACE2). The pediatric RP-GOs are fully susceptible to infection with SARS-CoV-2, where viral nucleoprotein is expressed in cells undergoing programmed cell death, while the efficiency of infection is significantly lower in fetal organoids. The RP-GOs derived from pediatric patients show sustained robust viral replication of SARS-CoV-2, compared with organoids derived from fetal stomachs. Transcriptomic analysis shows a moderate innate antiviral response and the lack of differentially expressed genes belonging to the interferon family. Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. However, the virus can efficiently infect gastric epithelium in pediatric patients, suggesting that the stomach might have an active role in fecal-oral transmission of SARS-CoV-2.

<h4>Background/aim</h4>This retrospective study aimed to investigate the clinical profile of pituitary insufficiency (PI) in adult population.<h4>Materials and methods</h4>One hundred and fifty patients who were diagnosed as having PI between 2012 and 2018 (53.3% female, mean age 48.13 ± 15.83 years) were retrospectively analyzed.<h4>Results</h4>Age at diagnosis was higher in females as compared with males (51.13 ± 15.95 vs. 44.70 ± 15.08 years, P = 0.012). The most frequent presenting signs were headache (29.4%) and visual disturbance (19.6%) in general. Females frequently presented with headache (33.3%), whereas males presented with sexual dysfunction (34.4%). The most frequent cause of PI was nonfunctional pituitary adenoma (28.8%) in general population. A frequent cause of PI was Sheehan’s syndrome (33.8%) among females and nonfunctional pituitary adenoma (38.6%) among males. Pituitary macroadenoma (75.8%) was frequent in pituitary tumors with PI. 55.3 % of the patients had 4 pituitary hormones deficiencies and 26.0% of patients had 3 pituitary hormones deficiencies. Gonadotropin deficiency was the leading pituitary hormone deficiency. The frequency of posttraumatic PI was 4.7% in the general population.<h4>Conclusion</h4>Nonfunctional pituitary adenoma was the most common cause of PI among males and Sheehan’s syndrome was a major etiologic factor in females. Sheehan’s syndrome remains an important health problem in Turkey although obstetric care has improved. Posttraumatic PI should be considered in the differential diagnosis of idiopathic PI.

Despite significant efforts to improve glioblastoma multiforme (GBM) treatment, GBM remains one of the most lethal cancers. Effective GBM treatments require sensitive intraoperative tumor visualization and effective postoperative chemotherapeutic delivery. Unfortunately, the diffusive and infiltrating nature of GBM limits the detection of GBM tumors, and current intraoperative visualization methods limit complete tumor resection. In addition, although chemotherapy is often used to eliminate any cancerous tissue remaining after surgery, most chemotherapeutic drugs do not effectively cross the brain-blood barrier (BBB) or enter GBM tumors. As a result, GBM has limited treatment options with high recurrence rates, and methods that improve its complete visualization during surgery and treatment are needed. Herein, we report a fluorescent nanoparticle platform for the near-infrared fluorescence (NIRF)-based tumor boundary visualization and image-guided drug delivery into GBM tumors. Our nanoplatform is based on ferumoxytol (FMX), an FDA-approved magnetic resonance imaging-sensitive superparamagnetic iron oxide nanoparticle, which is conjugated with hepthamethine cyanine (HMC), a NIRF ligand that specifically targets the organic anion transporter polypeptides that are overexpressed in GBM. We have shown that HMC-FMX nanoparticles cross the BBB and selectively accumulate in the tumor using orthotopic GBM mouse models, enabling NIRF-based visualization of infiltrating tumor tissue. In addition, HMC-FMX can encapsulate chemotherapeutic drugs, such as paclitaxel or cisplatin, and deliver these agents into GBM tumors, reducing tumor size and increasing survival. Taken together, these observations indicate that HMC-FMX is a promising nanoprobe for GBM surgical visualization and drug delivery.

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A-related leukodystrophy, Peroxisomal biogenesis disorders, POLR3-related Leukodystrophy, Vanishing White Matter, and Pelizaeus-Merzbacher Disease. Despite the relative frequency of these conditions, carrier-screening laboratories regularly test only 20 of the 55 leukodystrophy-related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.

Glioblastomas (GBMs) are incurable brain tumors with a high degree of cellular heterogeneity and genetic mutations. Transcription factors that normally regulate neural progenitors and glial development are aberrantly coexpressed in GBM, conferring cancer stem-like properties to drive tumor progression and therapeutic resistance. However, the functional role of individual transcription factors in GBMs in vivo remains elusive. Here, we demonstrate that the basic-helix-loop-helix transcription factor ASCL1 regulates transcriptional targets that are central to GBM development, including neural stem cell and glial transcription factors, oncogenic signaling molecules, chromatin modifying genes, and cell cycle and mitotic genes. We also show that the loss of ASCL1 significantly reduces the proliferation of GBMs induced in the brain of a genetically relevant glioma mouse model, resulting in extended survival times. RNA-seq analysis of mouse GBM tumors reveal that the loss of ASCL1 is associated with downregulation of cell cycle genes, illustrating an important role for ASCL1 in controlling the proliferation of GBM.

<h4>Introduction</h4>Variability exists in the disease trajectories of Alzheimer's disease (AD) patients. We performed a genome-wide association study to examine rate of cognitive decline (ROD) in patients with AD.<h4>Methods</h4>We tested for interactions between genetic variants and time since diagnosis to predict the ROD of a composite cognitive score in 3946 AD cases and performed pathway analysis on the top genes.<h4>Results</h4>Suggestive associations (P < 1.0 × 10^-6 ) were observed on chromosome 15 in DNA polymerase-γ (rs3176205, P = 1.11 × 10^-7 ), chromosome 7 (rs60465337,P = 4.06 × 10^-7 ) in contactin-associated protein-2, in RP11-384F7.1 on chromosome 3 (rs28853947, P = 5.93 × 10^-7 ), family with sequence similarity 214 member-A on chromosome 15 (rs2899492, P = 5.94 × 10^-7 ), and intergenic regions on chromosomes 16 (rs4949142, P = 4.02 × 10^-7 ) and 4 (rs1304013, P = 7.73 × 10^-7 ). Significant pathways involving neuronal development and function, apoptosis, memory, and inflammation were identified.<h4>Discussion</h4>Pathways related to AD, intelligence, and neurological function determine AD progression, while previously identified AD risk variants, including the apolipoprotein (APOE) ε4 and ε2 variants, do not have a major impact.

<h4>Background</h4>Splicing is crucial for proper gene expression, and is predominately executed by the major spliceosome. Conversely, 722 introns in 699 human minor intron-containing genes (MIGs) are spliced by the minor spliceosome. Splicing of these minor introns is disrupted in diseases caused by pathogenic variants in the minor spliceosome, ultimately leading to the aberrant expression of a subset of these MIGs. However, the effect of variants in minor introns and MIGs on diseases remains unexplored.<h4>Methods</h4>Variants in MIGs and associated clinical manifestations were identified using ClinVar. The HPO database was then used to curate the related symptoms and affected organ systems.<h4>Results</h4>We found pathogenic variants in 211 MIGs, which commonly resulted in intellectual disability, seizures and microcephaly. This revealed a subset of MIGs whose aberrant splicing may contribute to the pathogenesis of minor spliceosome-related diseases. Moreover, we identified 51 pathogenic variants in minor intron splice sites that reduce the splice site strength and can induce alternative splicing.<h4>Conclusion</h4>These findings highlight that disrupted minor intron splicing has a broader impact on human diseases than previously appreciated. The hope is that this knowledge will aid in the development of therapeutic strategies that incorporate the minor intron splicing pathway.

Chondrocytes, comparable to many cells from the connective tissue, dedifferentiate and end up in a similar fibroblastoid cell type, marked by the loss of the specific expression pattern. Here, chondrocytes isolated from osteoarthritic (OA) patients were investigated. The OA chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of known cartilage markers, such as SOX5, SOX6, SOX9, aggrecan and proteoglycan 4, remained unchanged. Since chondrocytes from OA and healthy tissue show different DNA methylation patterns, the underlying mechanisms of cartilage marker maintenance were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of dedifferentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2´-deoxycytidine (5-aza-dC) displayed no considerable impact on the maintenance of marker gene expression observed in the dedifferentiated state, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment for four weeks with 1 μM 5-aza-dC. In conclusion, 5-aza-dC affects the differentiation capacity of OA chondrocytes, while the global DNA methylation level remains stable. Furthermore, dedifferentiated chondrocytes isolated from late-stage OA patients represent a reliable cell source for in vitro studies and disease models without the need for additional alterations.

In the BACHD mouse model of Huntington's disease (HD), deletion of the N17 domain of the Huntingtin gene (BACHDΔN17, Q97) has been reported to lead to nuclear accumulation of mHTT and exacerbation of motor deficits, neuroinflammation and striatal atrophy (Gu et al., 2015). Here we characterized the effect of N17 deletion on dorsolateral striatal medium spiny neurons (MSNs) in BACHDΔN17 (Q97) and BACWTΔN17 (Q31) mice by comparing them to MSNs in wildtype (WT) mice. Mice were characterized on a series of motor tasks and subsequently whole cell patch clamp recordings with simultaneous biocytin filling of MSNs in in vitro striatal slices from these mice were used to comprehensively assess their physiological and morphological features. Key findings include that: Q97 mice exhibit impaired gait and righting reflexes but normal tail suspension reflexes and normal coats while Q31 mice do not differ from WT; intrinsic membrane and action potential properties are altered -but differentially so- in MSNs from Q97 and from Q31 mice; excitatory and inhibitory synaptic currents exhibit higher amplitudes in Q31 but not Q97 MSNs, while excitatory synaptic currents occur at lower frequency in Q97 than in WT and Q31 MSNs; there is a reduced total dendritic length in Q31 -but not Q97- MSNs compared to WT, while spine density and number did not differ in MSNs in the three groups. The findings that Q31 MSNs differed from Q97 and WT neurons with regard to some physiological features and structurally suggest a novel role of the N17 domain in the function of WT Htt. The motor phenotype seen in Q97 mice was less robust than that reported in an earlier study (Gu et al., 2015), and the alterations to MSN physiological properties were largely consistent with changes reported previously in a number of other mouse models of HD. Together this study indicates that N17 plays a role in the modulation of the properties of MSNs in both mHtt and WT-Htt mice, but does not markedly exacerbate HD-like pathogenesis in the BACHD model.

BACKGROUND Sepsis is an extremely common health issue with a considerable mortality rate in children. Our understanding about the pathogenic mechanisms of sepsis is limited. The aim of this study was to identify the differential expression genes (DEGs) in pediatric sepsis through comprehensive analysis, and to provide specific insights for the clinical sepsis therapies in children. MATERIAL AND METHODS Three pediatric gene expression profiles (GSE25504, GSE26378, GSE26440) were downloaded from the Gene Expression Omnibus (GEO) database. The difference expression genes (DEGs) between pediatric sepsis and normal control group were screened with the GEO2R online tool. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs were performed. Cytoscape with CytoHubba were used to identify the hub genes. Finally, NetworkAnalyst was used to construct the targeted microRNAs (miRNAs) of the hub genes. RESULTS Totally, 160 overlapping upward genes and 61 downward genes were identified. In addition, 5 KEGG pathways, including hematopoietic cell lineage, Staphylococcus aureus infection, starch and sucrose metabolism, osteoclast differentiation, and tumor necrosis factor (TNF) signaling pathway, were significantly enriched using a database for labeling, visualization, and synthetic discovery. In combination with the results of the protein-protein interaction (PPI) network and CytoHubba, 9 hub genes including ITGAM, TLR8, IL1ß, MMP9, MPO, FPR2, ELANE, SPI1, and C3AR1 were selected. Combined with DEG-miRNAs visualization, 5 miRNAs, including has-miR-204-5p, has-miR-211-5p, has-miR-590-5p, and has-miR-21-5p, were predicted as possibly the key miRNAs. CONCLUSIONS Our findings will contribute to identification of potential biomarkers and novel strategies for pediatric sepsis treatment.

<h4>Background</h4>ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown.<h4>Methods</h4>To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways.<h4>Results</h4>ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine¹¹⁰¹ in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27^S82 and eIF2a^S51, and triggers autophagosome formation in lung cancer cells.<h4>Conclusions</h4>These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.

Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1-RGMB-GDF5 assembly. Surprisingly, we show that all three RGMs inhibit GDF5 signaling, which is in stark contrast to RGM-mediated enhancement of signaling observed for other BMPs, like BMP2. Despite their opposite effect on GDF5 signaling, RGMs occupy the BMP type 1 receptor binding site similar to the observed interactions in RGM-BMP2 complexes. In the NEO1-RGMB-GDF5 complex, RGMB physically bridges NEO1 and GDF5, suggesting cross-talk between the GDF5 and NEO1 signaling pathways. Our crystal structures, combined with structure-guided mutagenesis of RGMs and BMP ligands, binding studies, and cellular assays suggest that RGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors. While our crystal structure analysis and in vitro binding data initially pointed towards a simple competition mechanism between RGMs and type 1 receptors as a possible basis for RGM-mediated GDF5 inhibition, further experiments utilizing BMP2-mimicking GDF5 variants clearly indicate a more complex mechanism that explains how RGMs can act as a functionality-changing switch for two structurally and biochemically similar signaling molecules.

Oncogenic RAS mutations are associated with DNA methylation changes that alter gene expression to drive cancer. Recent studies suggest that DNA methylation changes may be stochastic in nature, while other groups propose distinct signaling pathways responsible for aberrant methylation. Better understanding of DNA methylation events associated with oncogenic KRAS expression could enhance therapeutic approaches. Here we analyzed the basal CpG methylation of 11 KRAS-mutant and dependent pancreatic cancer cell lines and observed strikingly similar methylation patterns. KRAS knockdown resulted in unique methylation changes with limited overlap between each cell line. In KRAS-mutant Pa16C pancreatic cancer cells, while KRAS knockdown resulted in over 8,000 differentially methylated (DM) CpGs, treatment with the ERK1/2-selective inhibitor SCH772984 showed less than 40 DM CpGs, suggesting that ERK is not a broadly active driver of KRAS-associated DNA methylation. KRAS G12V overexpression in an isogenic lung model reveals >50,600 DM CpGs compared to non-transformed controls. In lung and pancreatic cells, gene ontology analyses of DM promoters show an enrichment for genes involved in differentiation and development. Taken all together, KRAS-mediated DNA methylation are stochastic and independent of canonical downstream effector signaling. These epigenetically altered genes associated with KRAS expression could represent potential therapeutic targets in KRAS-driven cancer.

<h4>Aims</h4>The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network initiated a second observational cohort study-the Symptom Patterns Study (SPS)-to further investigate the underlying pathophysiology of Urologic Chronic Pelvic Pain Syndrome (UCPPS) and to discover factors associated with longitudinal symptom changes and responses to treatments.<h4>Methods</h4>This multisite cohort study of males and females with UCPPS features a run-in period of four weekly web-based symptom assessments before a baseline visit, followed by quarterly assessments up to 36 months. Controls were also recruited and assessed at baseline and 6 months. Extensive clinical data assessing urological symptoms, nonurological pain, chronic overlapping pain syndromes, and psychosocial factors were collected. Diverse biospecimens for biomarker and microbiome studies, quantitative sensory testing (QST) data under multiple stimuli, and structural and functional neuroimaging scans were obtained under a standardized protocol.<h4>Results</h4>Recruitment was initiated (July 2015) and completed (February 2019) at six discovery sites. A total of 620 males and females with UCPPS and 73 Controls were enrolled, including 83 UCPPS participants who re-enrolled from the first MAPP Network cohort study (2009-2012). Baseline neuroimaging scans, QST measures, and biospecimens were obtained on 578 UCPPS participants. The longitudinal follow-up of the cohort is ongoing.<h4>Conclusions</h4>This comprehensive characterization of a large UCPPS cohort with extended follow-up greatly expands upon earlier MAPP Network studies and provides unprecedented opportunities to increase our understanding of UCPPS pathophysiology, factors associated with symptom change, clinically relevant patient phenotypes, and novel targets for future interventions.

The bone morphogenetic protein (BMP)-SMAD signaling pathway plays a central role in regulating hepcidin, which is the master hormone governing systemic iron homeostasis. Hepcidin is produced by the liver and acts on the iron exporter ferroportin to control iron absorption from the diet and iron release from body stores, thereby providing adequate iron for red blood cell production, while limiting the toxic effects of excess iron. BMP6 and BMP2 ligands produced by liver endothelial cells bind to BMP receptors and the coreceptor hemojuvelin (HJV) on hepatocytes to activate SMAD1/5/8 signaling, which directly upregulates hepcidin transcription. Most major signals that influence hepcidin production, including iron, erythropoietic drive, and inflammation, intersect with the BMP-SMAD pathway to regulate hepcidin transcription. Mutation or inactivation of BMP ligands, BMP receptors, HJV, SMADs or other proteins that modulate the BMP-SMAD pathway result in hepcidin dysregulation, leading to iron-related disorders, such as hemochromatosis and iron refractory iron deficiency anemia. Pharmacologic modulators of the BMP-SMAD pathway have shown efficacy in pre-clinical models to regulate hepcidin expression and treat iron-related disorders. This review will discuss recent insights into the role of the BMP-SMAD pathway in regulating hepcidin to control systemic iron homeostasis.

Histones are the main structural proteins of eukaryotic chromatin. Histone acetylation/ deacetylation are the epigenetic mechanisms of the regulation of gene expression and are catalyzed by histone acetyltransferases (HAT) and histone deacetylases (HDAC). These epigenetic alterations of DNA structure influence the action of transcription factors which can induce or repress gene transcription. The HATs catalyze acetylation and the events related to gene transcription and are also responsible for transporting newly synthesized histones from the cytoplasm to the nucleus. The activity of HDACs is mainly involved in silencing gene expression and according to their specialized functions are divided into classes I, II, III and IV. The disturbance of the expression and mutations of HDAC genes causes the aberrant transcription of key genes regulating important cancer pathways such as cell proliferation, cell-cycle regulation and apoptosis. In view of their role in cancer pathways, HDACs are considered promising therapeutic targets and the development of HDAC inhibitors is a hot topic in the search for new anticancer drugs. The present review will focus on HDACs I, II and IV, the best known inhibitors and potential alternative inhibitors derived from natural and synthetic products which can be used to influence HDAC activity and the development of new cancer therapies.

The signaling pathway of the microtubule-associated protein kinase or extracellular regulated kinase (MAPK/ERK) is a common mechanism of extracellular information transduction from extracellular stimuli to the intracellular space. The transduction of information leads to changes in the ongoing metabolic pathways and the modification of gene expression patterns. In the central nervous system, ERK is expressed ubiquitously, both temporally and spatially. As for the temporal ubiquity, this signaling system participates in three key moments: (i) Embryonic development; (ii) the early postnatal period; and iii) adulthood. During embryonic development, the system is partly responsible for the patterning of segmentation in the encephalic vesicle through the FGF8-ERK pathway. In addition, during this period, ERK directs neurogenesis migration and the final fate of neural progenitors. During the early postnatal period, ERK participates in the maturation process of dendritic trees and synaptogenesis. During adulthood, ERK participates in social and emotional behavior and memory processes, including long-term potentiation. Alterations in mechanisms related to ERK are associated with different pathological outcomes. Genetic alterations in any component of the ERK pathway result in pathologies associated with neural crest derivatives and mental dysfunctions associated with autism spectrum disorders. The MAP-ERK pathway is a key element of the neuroinflammatory pathway triggered by glial cells during the development of neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as prionic diseases. The process triggered by MAPK/ERK activation depends on the stage of development (mature or senescence), the type of cellular element in which the pathway is activated, and the anatomic neural structure. However, extensive gaps exist with regards to the targets of the phosphorylated ERK in many of these processes.

<h4>Objective</h4>To compare the effect of delayed cord clamping at 30 seconds and 1 minute on the incidence of neonatal hematocrit, anemia, maternal and neonatal complications in term cesarean delivered neonates.<h4>Methods</h4>An opened labelled, randomized controlled trial was undertaken. The 160 healthy term cesarean-born neonates were randomly allocated to either 30 seconds or 1-minute groups of delayed cord clamping (DCC) (groups 1 and 2). Neonatal venous hematocrit (Hct) and microbilirubin (Mb) were measured at 48-72 hours after birth.<h4>Results</h4>One hundred and fifty-nine neonates completed this study. Mean neonatal hematocrit ± standard deviation at 48-72 hours was 49.9 ± 6.0% in group 1 and 51.2 ± 5.9% in group 2 without a statistical difference. Neonatal anemia (Hct less than 45%) occurred in 14/79 neonates (17.7%) in group 1 and in 8/80 cases (10.0%) in group 2 without a significant difference between groups. The incidence of neonatal jaundice and polycythemia (hematocrit more than 65%) was similar between groups. There were no significant differences; in the estimated blood loss during the operation, the incidence of postpartum hemorrhage and other maternal and neonatal complications.<h4>Conclusion</h4>Neonatal hematocrit was not significantly different following DCC at 30 seconds and at 1 minute, but the incidence of neonatal anemia decreased with the longer timing of DCC. The estimated blood loss and other complications were not different between the two groups. Therefore, one minute-DCC should be considered for neonatal anemic prevention when compared with 30 seconds-DCC.

Neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis, are characterized by a progressive loss of selective neuron subtypes in the central nervous system (CNS). Although various factors account for the initiation and development of these diseases, accumulating evidence shows that impaired mitochondrial function is a prominent and common mechanism. Mitochondria play a critical role in neurons and are involved in energy production, cellular metabolism regulation, intracellular calcium homeostasis, immune responses, and cell fate. Thus, cells in the CNS heavily rely on mitochondrial integrity. Many aspects of mitochondrial dysfunction are manifested in neurodegenerative diseases, including aberrant mitochondrial quality control (mitoQC), mitochondrial-driven inflammation, and bioenergetic defects. Herein, we briefly summarize the molecular basis of mitoQC, including mitochondrial proteostasis, biogenesis, dynamics, and organelle degradation. We also focus on the research, to date, regarding aberrant mitoQC and mitochondrial-driven inflammation in several common neurodegenerative diseases. In addition, we outline novel therapeutic strategies that target aberrant mitoQC in neurodegenerative diseases.

Plasmids can provide advantageous traits to host bacteria, although they may impose a fitness cost. Chromosome-encoded factors are important for regulating the expression of genes on plasmids, and host chromosomes may differ in terms of their interactions with a given plasmid. Accordingly, differences in fitness cost loading and compensatory co-evolution may occur for various host chromosome/plasmid combinations. However, the mechanisms of compensatory evolution are highly divergent and require further insights. Here, we reveal novel evolutionally mechanisms of <i>Pseudomonas putida</i> KT2440 to improve the fitness cost imposed by the incompatibility P-1 (IncP-1) multidrug resistance plasmid RP4. A mixed culture of RP4-harboring and -free KT2440 cells was serially transferred every 24 h under non-selective conditions. Initially, the proportion of RP4-harboring cells decreased rapidly, but it immediately recovered, suggesting that the fitness of RP4-harboring strains improved during cultivation. Larger-sized colonies appeared during 144-h mixed culture, and evolved strains isolated from larger-sized colonies showed higher growth rates and fitness than those of the ancestral strain. Whole-genome sequencing revealed that evolved strains had one of two mutations in the same intergenic region of the chromosome. Based on the research of another group, this region is predicted to contain a stress-inducible small RNA (sRNA). Identification of the transcriptional start site in this sRNA indicated that one mutation occurred within the sRNA region, whereas the other was in its promoter region. Quantitative reverse-transcription PCR showed that the expression of this sRNA was strongly induced by RP4 carriage in the ancestral strain but repressed in the evolved strains. When the sRNA region was overexpressed in the RP4-free strain, the fitness decreased, and the colony size became smaller. Using transcriptome analysis, we also showed that the genes involved in amino acid metabolism and stress responses were differentially transcribed by overexpression of the sRNA region. These results indicate that the RP4-inducible chromosomal sRNA was responsible for the fitness cost of RP4 on KT2440 cells, where this sRNA is of key importance in host evolution toward rapid amelioration of the cost.

Seizure patterns observed in patients with epilepsy suggest that circadian rhythms and sleep/wake mechanisms play some role in the disease. This review addresses key topics in the relationship between circadian rhythms and seizures in epilepsy. We present basic information on circadian biology, but focus on research studying the influence of both the time of day and the sleep/wake cycle as independent but related factors on the expression of seizures in epilepsy. We review studies investigating how seizures and epilepsy disrupt expression of core clock genes, and how disruption of clock mechanisms impacts seizures and the development of epilepsy. We focus on the overlap between mechanisms of circadian-associated changes in SCN neuronal excitability and mechanisms of epileptogenesis as a means of identifying key pathways and molecules that could represent new targets or strategies for epilepsy therapy. Finally, we review the concept of chronotherapy and provide a perspective regarding its application to patients with epilepsy based on their individual characteristics (i.e., being a "morning person" or a "night owl"). We conclude that better understanding of the relationship between circadian rhythms, neuronal excitability, and seizures will allow both the identification of new therapeutic targets for treating epilepsy as well as more effective treatment regimens using currently available pharmacological and non-pharmacological strategies.

Peroxisomes participate in essential cellular metabolic processes, such as oxidation of fatty acids (FAs) and maintenance of reactive oxygen species (ROS) homeostasis. Peroxisomes must communicate with surrounding organelles to exchange information and metabolites. The formation of membrane contact sites (MCSs), where protein-protein or protein-lipid complexes tether the opposing membranes of two organelles, represents an essential means of organelle crosstalk. Peroxisomal MCS (PO-MCS) studies are emerging but are still in the early stages. In this review, we summarize the identified PO-MCSs with the ER, mitochondria, lipid droplets, and lysosomes in mammalian cells and discuss their tethering mechanisms and physiological roles. We also highlight several features of PO-MCSs that may help future studies.

<h4>Background and purpose</h4>Ischemia-reperfusion injuries (IRIs) can aggravate the condition of some patients with acute occlusion of major intracranial artery (AOMIA) who received endovascular thrombectomy. Here, we provided data confirming the association of Repressor Element-1 Silencing Transcription factor (REST) with the long-term neuroprotective effect of the middle cerebral artery occlusion (MCAO) rats underwent Gradual Flow Restoration (GFR).<h4>Methods</h4>Long term neuroprotective effects of GFR intervention were evaluated on MCAO rats model after 3d and 7d reperfusion. The neurological deficit score and TTC staining were performed to evaluate the degree of brain damage in GFR and other interventions at different time. Differentially expressed genes related to cerebral ischemia reperfusion injury (CIRI) were initially screened and identified using GSE32529 microarray analysis. REST protein expression in rat brain cortex infarction was detected by Western blot analysis.<h4>Results</h4>MCAO rats intervened with GFR exhibited reduced neurological deficit (P < 0.05) and alleviated brain infarction volume (P < 0.01). The REST gene with up-regulated expression and its downstream genes with down-regulated expression were screened by Microarray analysis. The brain cortex infarction in MCAO rats produced high levels of REST expression. The GFR intervention inhibited REST expression, and alleviated brain injury on MCAO rats.<h4>Conclusion</h4>Our results demonstrated that GFR intervention plays a long-term neuroprotective role and reduces brain edema and damage at reperfusion, possibly by inhibiting REST expression.

<h4>Purpose</h4>Microbiota-derived metabolites could alter the brain tissue toward the neurodegeneration disease. This study aims to select the genes associated with Propionic acid (PPA) and compromise Alzheimer's disease (AD) to find the possible roles of PPA in AD pathogenesis.<h4>Methods</h4>Microbiota-derived metabolites could alter the brain tissue toward the neurodegeneration disease. This study aims to select the genes associated with Propionic acid (PPA) and compromise Alzheimer's disease (AD) to find the possible roles of PPA in AD pathogenesis.<h4>Results</h4>Amongst all genes associated with PPA and AD, 284 genes to be shared by searching databases and were subjected to further analysis. AD-PPA genes mainly involved in cancer, bacterial and virus infection, and neurological and non-neurological diseases. Gene Ontology and pathway analysis covered the most AD hallmark, such as amyloid formation, apoptosis, proliferation, inflammation, and immune system. Network analysis revealed hub and bottleneck genes. MCODE analysis also indicated the seed genes represented in the significant subnetworks. <i>ICAM1</i> and <i>CCND1</i> were the hub, bottleneck, and seed genes.<h4>Conclusions</h4>PPA interacted genes implicated in AD act through pathways initiate neuronal cell death. In sum up, AD-PPA shared genes exhibited evidence that supports the idea PPA secreted from bacteria could alter brain physiology toward the emerging AD signs. This idea needs to confirm by more future investigation in animal models.

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivatives, short and extended derivatives, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration (MIC) against C. albicans strains. The short derivatives were more potent against the C. albicans strains (e. g., MIC 2-(4-chlorophenyl)-N-(2,4-dichlorobenzyl)-3-(1H-imidazol-1-yl)propanamide (5 f) <0.03 μg/mL, N-(4-((4-chlorophenyl)sulfonamido)benzyl)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propanamide (12 c), 1 μg/mL, fluconazole 0.125 μg/mL) but both displayed comparable enzyme binding and inhibition (5 f K_d 62±17 nM, IC₅₀ 0.46 μM; 12 c K_d 43±18 nM, IC₅₀ 0.33 μM, fluconazole K_d 41±13 nM, IC₅₀ 0.31 μM, posaconazole K_d 43±11 nM, IC₅₀ 0.2 μM). The short series had poor selectivity for CaCYP51 over the human homologue, whereas the selectivity of the extended series, for example, compound 12 c, was higher (21.5-fold) than posaconazole (4.7-fold) based on K_d values, although posaconazole was more selective (615-fold) than 12 c (461-fold) based on IC₅₀ values. Based on inhibitory activity and selectivity profile, the extended series are the better of the two series for further development.

<h4>Aims</h4>To investigate the anti-inflammatory activity of an invasive and Hp65-producing strain Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) in acute 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in mice as an innovative therapeutic strategy against Crohn's disease (CD).<h4>Methods and results</h4>The pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, the anti-inflammatory capacity of the recombinant strain was evaluated in colitis induced with TNBS in BALB/c mice. Oral administration of the recombinant strain was able to attenuated the severity of colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and secretory immunoglobulin A levels.<h4>Conclusions</h4>The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to a reduction in inflammatory damage in experimental CD.<h4>Significance and impact of the study</h4>This study, which used L. lactis for the production and delivery of Hsp65, has scientific relevance because it shows the efficacy of this new strategy based on therapeutic protein delivery into mammalian enterocytes.

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of <i>Pet1</i>-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing <i>Pet1</i> neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-<i>Pet1</i> neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that <i>P2ry1-Pet1</i> DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties. These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of <i>Pet1</i> neuron subtypes.

<h4>Background</h4>Umbilical cord clamping is a crucial step during the third stage of labour that separates the newborn from the placenta. Despite the available evidence that delayed umbilical cord clamping is more beneficial to infants, as well as the existence of 2014 WHO recommendation that the umbilical cord should be clamped between 1 and 3 minutes, its implementation is still low in many countries including Tanzania.<h4>Objective</h4>This study describes the experiences and perceptions of nurse-midwives`and obstetricians`about the timing of umbilical cord clamping at a regional referral hospital in Tanzania.<h4>Methods</h4>A descriptive qualitative study design that adopted a purposeful sampling strategy to recruit 19 participants was used. Nine semi-structured interviews with six nurse-midwives`and three obstetricians`, as well as one focus group discussion with ten nurse-midwives`were conducted. Thematic analysis guided the analysis of data.<h4>Results</h4>Three main themes generated from the data, each having 2 to 5 subthemes. 1. Experiences about the timing of umbilical cord clamping. 2. Perceptions about the umbilical cord clamping. 3. Factors influencing the practice of delayed umbilical cord clamping to improve newborn health outcomes.<h4>Conclusion</h4>Although the nurse-midwives`and obstetricians`commonly practiced clamping the umbilical cord immediately after delivery, they understood that delayed cord clamping has a potential benefit of oxygenation to the newborn in the event of the need for resuscitation. To move forward with the good practice in maternal and newborn care, proper pre-service and providers training on matters underlying childbirth is essential to address the gap of knowledge. Delayed cord clamping should be practiced widely to improve the health outcomes of the newborn.

<h4>Background</h4>Post-transcriptional methylation modifications, including 5-methylcytosine (m5C) modification, are closely related to the tumorigenesis of cancers. However, the mRNA profile of m5C modification in hepatocellular carcinoma (HCC) is unknown.<h4>Methods</h4>Methylated RNA immunoprecipitation sequencing was performed to identify m5C peaks on mRNA of human HCC tissues and adjacent tissues, and differences in m5C between the two groups were analyzed. In addition, we conducted a bioinformatics analysis to predict the function of specific methylated transcripts.<h4>Results</h4>We found that there was a noticeable difference in m5C between HCC and paired non-tumor tissues, suggesting that m5C could play a role in the pathogenesis of HCC. In addition, analyses of gene ontology and the Kyoto Encyclopedia of Genes and Genomes showed that the unique distribution pattern of mRNA m5C in HCC was associated with a wide range of cellular functions.<h4>Conclusions</h4>Our results revealed different distribution patterns of m5C in HCC and adjacent tissues and provided new insights into a novel function of m5C RNA methylation of mRNA in HCC progression.

<h4>Objectives</h4>The present review systematically summarized existing publications regarding the genetic associations between voltage-gated calcium channels (VGCCs) and autism spectrum disorder (ASD).<h4>Methods</h4>A comprehensive literature search was conducted to gather pertinent studies in three online databases. Two authors independently screened the included records based on the selection criteria. Discrepancies in each step were settled through discussions.<h4>Results</h4>From 1163 resulting searched articles, 28 were identified for inclusion. The most prominent among the VGCCs variants found in ASD were those falling within loci encoding the α subunits, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, and CACNA1I as well as those of their accessory subunits CACNB2, CACNA2D3, and CACNA2D4. Two signaling pathways, the IP3-Ca²⁺ pathway and the MAPK pathway, were identified as scaffolds that united genetic lesions into a consensus etiology of ASD.<h4>Conclusions</h4>Evidence generated from this review supports the role of VGCC genetic variants in the pathogenesis of ASD, making it a promising therapeutic target. Future research should focus on the specific mechanism that connects VGCC genetic variants to the complex ASD phenotype.

<h4>Background</h4>This study aimed to demonstrate that the material of the occlusal splint can be chosen according to the needs of individual patients and contribute to the knowledge of the wear rate of these materials.<h4>Methods</h4>In this study, four occlusal splint materials (Sr Ivocap Heat Cured, Valplast, SR Ivocap Elastomer and Eclipse) and three antagonists (natural tooth enamel, inCoris TZI and IPS e.max Press ceramic materials) were used. Each wear test was performed using a chewing simulator (n = 16; test load: 50 N; number of cycles: 10,000, 20,000 and 30,000; continuous rinsing with water at 30 °C for the wet condition). The Shapiro Wilk test was used for normal distribution suitability. Antagonist on average wear quantities both main effects and interactions of material, cycle and condition factors were investigated by Univariate variance analysis. Multiple comparisons were examined using the Games-Howell test.<h4>Results</h4>There was a statistically significant effect of the difference in materials on the amount of wear (p < 0.001). Furthermore, there was a statistically significant difference among the mean values of all materials (p > 0.001). The highest mean value was obtained with Eclipse (0,318 μm³), and the lowest mean value was obtained with Valplast (0,134 μm³).<h4>Conclusion</h4>Our study found differences in the in vitro wear rate among various occlusal splint materials.

The availability of MHC-binding prediction tools has been useful in guiding studies aimed at identifying candidate target Ags to generate reactive T cells and to characterize viral and tumor-reactive T cells. Nevertheless, prediction algorithms appear to function poorly for epitopes containing cysteine (Cys) residues, which can oxidize and form disulfide bonds with other Cys residues under oxidizing conditions, thus potentially interfering with their ability to bind to MHC molecules. Analysis of the results of HLA-A*02:01 class I binding assays carried out in the presence and absence of the reducing agent 2-ME indicated that the predicted affinity for 25% of Cys-containing epitopes was underestimated by a factor of 3 or more. Additional analyses were undertaken to evaluate the responses of human CD8⁺ tumor-reactive T cells against 10 Cys-containing HLA class I-restricted minimal determinants containing substitutions of α-aminobutyric acid (AABA), a cysteine analogue containing a methyl group in place of the sulfhydryl group present in Cys, for the native Cys residues. Substitutions of AABA for Cys at putative MHC anchor positions often significantly enhanced T cell recognition, whereas substitutions at non-MHC anchor positions were neutral, except for one epitope where this modification abolished T cell recognition. These findings demonstrate the need to evaluate MHC binding and T cell recognition of Cys-containing peptides under conditions that prevent Cys oxidation, and to adjust current prediction binding algorithms for HLA-A*02:01 and potentially additional class I alleles to more accurately rank peptides containing Cys anchor residues.

G protein-coupled receptors (GPCRs) represent the largest family of cell membrane proteins, with >800 GPCRs in humans alone, and recognize highly diverse ligands, ranging from photons to large protein molecules. Very important to human medicine, GPCRs are targeted by about 35% of prescription drugs. GPCRs are characterized by a seven-transmembrane α-helical structure, transmitting extracellular signals into cells to regulate major physiological processes via heterotrimeric G proteins and β-arrestins. Initially viewed as receptors whose signaling via G proteins is delimited to the plasma membrane, it is now recognized that GPCRs signal also at various intracellular locations, and the mechanisms and (patho)physiological relevance of such signaling modes are actively investigated. The propensity of GPCRs to adopt different signaling modes is largely encoded in the structural plasticity of the receptors themselves and of their signaling complexes. Here, we review emerging modes of GPCR signaling via endosomal membranes and the physiological implications of such signaling modes. We further summarize recent structural insights into mechanisms of GPCR activation and signaling. We particularly emphasize the structural mechanisms governing the continued GPCR signaling from endosomes and the structural aspects of the GPCR resensitization mechanism and discuss the recently uncovered and important roles of lipids in these processes.

Analyzing the associations between genotypic changes and phenotypic traits on a genome-wide scale can contribute to understanding the functional roles of distinct genetic variations during breed development. We performed a whole-genome analysis of Angus and Jersey cattle breeds using conditional mutual information, which is an information-theoretic method estimating the conditional independency among multiple factor variables. The proposed conditional mutual information-based approach allows breed-discriminative genetic variations to be explicitly identified from tens of millions of SNP (single nucleotide polymorphism) positions on a genome-wide scale while minimizing the usage of prior knowledge. Using this data-driven approach, we identified biologically relevant functional genes, including breed-specific variants for cattle traits such as beef and dairy production. The identified lipid-related genes were shown to be significantly associated with lipid and triglyceride metabolism, fat cell differentiation, and muscle development. In addition, we confirmed that milk-related genes are involved in mammary gland development, lactation, and mastitis-associated processes. Our results provide the distinct properties of Angus and Jersey cattle at a genome-wide level. Moreover, this study offers important insights into discovering unrevealed genetic variants for breed-specific traits and the identification of genetic signatures of diverse cattle breeds with respect to target breed-specific properties.

Horizontal gaze palsy with progressive scoliosis (HGPPS) is a rare, inherited disorder characterized by a congenital absence of conjugate horizontal eye movements with progressive scoliosis developing in childhood and adolescence. Mutations in the Roundabout (<i>ROBO</i>3) gene located on chromosome 11q23-25 are responsible for the development of horizontal gaze palsy and progressive scoliosis. However, some studies redefined the locus responsible for this pathology to a 9-cM region. This study carried out a systematic review in which 25 documents were analyzed, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards. The search was made in the following electronic databases from January 1995 to October 2019: PubMed, Scopus, Web of Science, PEDRO, SPORT Discus, and CINAHL. HGPPS requires a multidisciplinary diagnostic approach, in which magnetic resonance imaging might be the first technique to suggest the diagnosis, which should be verified by an analysis of the <i>ROBO</i>3 gene. This is important to allow for adequate ocular follow up, apply supportive therapies to prevent the rapid progression of scoliosis, and lead to appropriate genetic counseling.

Huntington's disease is a severe and currently incurable neurodegenerative disease. An autosomal dominant mutation in the Huntingtin gene (<i>HTT</i>) causes an increase in the polyglutamine fragment length at the protein N-terminus. The consequence of the mutation is the death of neurons, mostly striatal neurons, leading to the occurrence of a complex of motor, cognitive and emotional-volitional personality sphere disorders in carriers. Despite intensive studies, the functions of both mutant and wild-type huntingtin remain poorly understood. Surprisingly, there is the selective effect of the mutant form of HTT even on nervous tissue, whereas the protein is expressed ubiquitously. Huntingtin plays a role in cell physiology and affects cell transport, endocytosis, protein degradation and other cellular and molecular processes. Our experimental data mining let us conclude that a significant part of the Huntingtin-involved cellular processes is mediated by microtubules and other cytoskeletal cell structures. The review attempts to look at unresolved issues in the study of the huntingtin and its mutant form, including their functions affecting microtubules and other components of the cell cytoskeleton.

Regenerative properties are different in every human tissue. Nowadays, with the increasing popularity of dental implants, bone regenerative procedures called augmentations are sometimes crucial in order to perform a successful dental procedure. Tissue engineering allows for controlled growth of alveolar and periodontal tissues, with use of scaffolds, cells, and signalling molecules. By modulating the patient's tissues, it can positively influence poor integration and healing, resulting in repeated implant surgeries. Application of nanomaterials and stem cells in tissue regeneration is a newly developing field, with great potential for maxillofacial bony defects. Nanostructured scaffolds provide a closer structural support with natural bone, while stem cells allow bony tissue regeneration in places when a certain volume of bone is crucial to perform a successful implantation. Several types of selected nanomaterials and stem cells were discussed in this study. Their use has a high impact on the efficacy of the current and future procedures, which are still challenging for medicine. There are many factors that can influence the regenerative process, while its general complexity makes the whole process even harder to control. The aim of this study was to evaluate the effectiveness and advantage of both stem cells and nanomaterials in order to better understand their function in regeneration of bone tissue in oral cavity.

Members of the tumour necrosis factor (TNF) superfamily OX40L and CD70 and their receptors are costimulating signalling axes critical for adequate T cell activation in humans and mice but characterisation of these molecules in other species including ruminants is lacking. Here we cloned and expressed the predicted ovine orthologues of the receptors OX40 and CD27, as well as soluble recombinant forms of their potential ovine ligands, <i>Oa</i>OX40L and <i>Oa</i>CD70. Using biochemical and immunofluorescence analyses, we show that both signalling axes are functional in sheep. We show that oligomeric recombinant ligand constructs are able to induce signalling through their receptors on transfected cells. Recombinant defective human adenoviruses were constructed to express the soluble forms of <i>Oa</i>OX40L and <i>Oa</i>CD70. Both proteins were detected in the supernatant of adenovirus-infected cells and shown to activate NF-κB signalling pathway through their cognate receptor. These adenovirus-secreted <i>Oa</i>OX40L and <i>Oa</i>CD70 forms could also activate ovine T cell proliferation and enhance IFN-γ production in CD4⁺ and CD8⁺ T cells. Altogether, this study provides the first characterisation of the ovine costimulatory OX40L-OX40 and CD70-CD27 signalling axes, and indicates that their activation in vivo may be useful to enhance vaccination-induced immune responses in sheep and other ruminants.

The use of multiphase flows in microfluidics to carry dispersed phase material (droplets, particles, bubbles, or fibers) has many applications. In this review paper, we focus on such flows on centrifugal microfluidic platforms and present different methods of dispersed phase material generation. These methods are classified into three specific categories, i.e., step emulsification, crossflow, and dispenser nozzle. Previous works on these topics are discussed and related parameters and specifications, including the size, material, production rate, and rotational speed are explicitly mentioned. In addition, the associated theories and important dimensionless numbers are presented. Finally, we discuss the commercialization of these devices and show a comparison to unveil the pros and cons of the different methods so that researchers can select the centrifugal droplet/particle generation method which better suits their needs.

Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.

Thietanes are important aliphatic four-membered thiaheterocycles that are found in the pharmaceutical core and structural motifs of some biological compounds. They are also useful intermediates in organic synthesis. Various synthetic methods of thietanes have been developed, including inter- and intramolecular nucleophilic thioetherifications, photochemical [2 + 2] cycloadditions, ring expansions and contractions, nucleophilic cyclizations, and some miscellaneous methods. The recently developed methods provide some new strategies for the efficient preparation of thietanes and their derivatives. This review focuses on the synthetic methods to construct thietane backbones developed during 1966 to 2019.

Idiopathic nephrotic syndrome (INS) is a disease involving injury to podocytes in the glomerular filtration barrier, and its specific causes have not been elucidated. Transfer RNA-derived fragments (tRFs), products of precise tRNA cleavage, have been indicated to play critical roles in various diseases. Currently, there is no relevant research on the role of tRFs in INS. This study intends to explore the changes in and importance of tRFs during podocyte injury in vitro and to further analyze the potential mechanism of INS. Differentially expressed tRFs in the adriamycin-treated group were identified by high-throughput sequencing and further verified by quantitative RT-PCR. In total, 203 tRFs with significant differential expression were identified, namely, 102 upregulated tRFs and 101 downregulated tRFs (<i>q</i> < 0.05, ∣log2FC | ≥2). In particular, AS-tDR-008924, AS-tDR-011690, tDR-003634, AS-tDR-013354, tDR-011031, AS-tDR-001008, and AS-tDR-007319 were predicted to be involved in podocyte injury by targeting the Gpr, Wnt, Rac1, and other genes. Furthermore, gene ontology analysis showed that these differential tRFs were strongly associated with podocyte injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton, and insulin-activate receptor activity. KEGG pathway analysis predicted that they participated in the PI3K-Akt signaling pathway, Wnt signaling pathway, and Ras signaling pathway. It was reported that these pathways contribute to podocyte injury. In conclusion, our study revealed that changes in the expression levels of tRFs might be involved in INS. Seven of the differentially expressed tRFs might play important roles in the process of podocyte injury and are worthy of further study.

2-Hydroxy-4<i>H</i>-quinolizin-4-one scaffolds were enzymatically synthesized by integrating three enzymes including phenylacetate-CoA ligase (PcPCL) from an endophytic fungus <i>Penicillium chrysogenum</i> MT-12, malonyl-CoA synthase (AtMatB) from <i>Arabidopsis thaliana</i>, and a type III polyketide synthase (HsPKS3) from Chinese club moss <i>Huperzia serrata</i>. The findings paved the way to produce these kinds of structurally interesting alkaloids by engineered microorganisms.

The MeOH/CH₂Cl₂ (1:1) extracts of the roots and leaves of <i>Beilschmiedia louisii</i> and <i>B. obscura</i> showed potent antitrypanosomal activity during preliminary screening on <i>Trypanosoma brucei brucei</i>. Phytochemical investigation of these extracts led to the isolation of a mixture of two new endiandric acid derivatives beilschmiedol B (<b>1</b>) and beilschmiedol C (<b>2</b>), and one new phenylalkene obscurene A (<b>3</b>) together with twelve known compounds (<b>4</b>-<b>15</b>). In addition, four new derivatives (<b>11a</b>-<b>11d</b>) were synthesized from compound <b>11</b>. Their structures were elucidated based on their NMR and MS data. Compounds <b>5</b>, <b>6</b>, and <b>7</b> were isolated for the first time from the <i>Beilschmiedia</i> genus. Additionally, the NMR data of compound <b>4</b> are given here for the first time. The isolates were evaluated for their antitrypanosomal and antimalarial activities against <i>Tb brucei</i> and the <i>Plasmodium falciparum</i> chloroquine-resistant strain <i>Pf3D7</i> in vitro, respectively. From the tested compounds, the mixture of new compounds <b>1</b> and <b>2</b> exhibited the most potent antitrypanosomal activity in vitro with IC₅₀ value of 4.91 μM.

Immune checkpoint inhibitors (ICIs) have changed therapeutic algorithms in several malignancies, although intrinsic and secondary resistance is still an issue. In this context, the dysregulation of immuno-metabolism plays a leading role both in the tumor microenvironment (TME) and at the host level. In this review, we summarize the most important immune-metabolic factors and how they could be exploited therapeutically. At the cellular level, an increased concentration of extracellular adenosine as well as the depletion of tryptophan and uncontrolled activation of the PI3K/AKT pathway induces an immune-tolerant TME, reducing the response to ICIs. Moreover, aberrant angiogenesis induces a hypoxic environment by recruiting VEGF, T_reg cells and immune-suppressive tumor associated macrophages (TAMs). On the other hand, factors such as gender and body mass index seem to affect the response to ICIs, while the microbiome composition (and its alterations) modulates both the response and the development of immune-related adverse events. Exploiting these complex mechanisms is the next goal in immunotherapy. The most successful strategy to date has been the combination of antiangiogenic drugs and ICIs, which prolonged the survival of patients with non-small-cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC), while results from tryptophan pathway inhibition studies are inconclusive. New exciting strategies include targeting the adenosine pathway, TAMs and the microbiota with fecal microbiome transplantation.

<h4>Background</h4>The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result.<h4>Aim</h4>This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection.<h4>Methods</h4>Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells.<h4>Results</h4>The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study.<h4>Conclusion</h4>A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences, particularly for epidemiological surveillance of ASF.

<h4>SUMMARY</h4> 5-Aza-2′-deoxycytidine, also known as decitabine, is a DNA-methyltransferase inhibitor (DNMTi) used to treat acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERV), which can induce an immune response by acting as cellular double-stranded RNAs (dsRNAs). Here, we employ an image-based screening platform to identify dsRNA-binding factors that mediate the downstream effect of ERV induction. We find that Staufen1 (Stau1) knockdown decreases the interferon signature and rescues decitabine-mediated cell death. Moreover, Stau1 directly binds to ERV RNAs and stabilizes them, together with a long non-coding RNA TINCR. We further show that TINCR enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that AML patients with low Stau1 and TINCR expressions exhibits inferior treatment outcomes to the DNMTi therapy. Our study reveals that decitabine-mediated cell death is a consequence of complex interactions among different dsRNA-binding proteins for access to their common dsRNA targets. <h4>HIGHLIHGTS</h4> Image-based RNAi screening reveals multiple dsRBPs regulate response to decitabine Stau1 binds to ERV RNAs and affects their stability and subcellular localization TINCR binds to Stau1 and enhances Stau1-ERV interactions AML/MDS patients with low Stau1 and TINCR expressions show poor response to DNMTis

We performed a genome-wide association study to investigate the association between single nucleotide polymorphisms and anthracycline-induced cardiotoxicity (ACT) in patients diagnosed with early breast cancer. From January 2000 to December 2015, 8490 patients underwent breast surgery at the National Cancer Center in Korea. Patients who received doxorubicin (cumulative dose 240 mg/m² -300 mg/m² ) with or without trastuzumab as a neoadjuvant/adjuvant therapy were included in our cohort. Sixty-seven patients in our cohort were diagnosed with ACT. Clinical data, including age, body weight, height, cancer stage, trastuzumab treatment, comorbidities, and concomitant medications, were collected retrospectively. Patients were classified as having either persistent or transient ACT based on their clinical course. In total, 346 946 single nucleotide polymorphisms in 42 cases and 215 controls were tested in this study. Body mass index (BMI) ≥25 kg/m² [odds ratio (OR) = 2.45, 95% confidence interval (CI), 1.23-4.88, P = .011] and trastuzumab use (OR = 2.40, 95% CI, 1.11-5.17, P = .026) were identified as significant risk factors. We found 7 genetic variants for ACT including rs17530621 (SHISA3, P = 3.10E-06), rs11894115 (MPP4, P = 4.71E-06), rs58328254 (RPL7, P = 6.09E-06), and rs117299725 (PRUNE2, P = 8.53E-06), although none of these variants reached the Bonferroni-corrected significance level when adjusted for BMI and trastuzumab use ( = α1.44E-07 based on 0.05/346 946). rs117299725 was a common variant when only the persistent ACT group was analyzed separately. It is meaningful that our study analyzed comprehensively the influence of genetic variation on ACT, along with some clinical factors in Asian breast cancer patients who received anthracycline with or without trastuzumab. Further research will be needed on candidate genetic variants found in this study.

<h4>Background</h4>Bariatric surgery can effectively treat morbid obesity; however, micronutrient deficiencies are common despite recommendations for high-dose supplements. Genetic predisposition to deficiencies underscores necessary identification of high-risk candidates. Personalized nutrition (PN) can be a tool to manage these deficiencies.<h4>Methods</h4>Medline, PubMed, and Google Scholar were searched. Articles involving genetic testing, micronutrient metabolism, and bariatric surgery were included.<h4>Results</h4>Studies show associations between genetic variants and micronutrient metabolism. Research demonstrates genetic testing to be a predictor for outcomes among obesity and bariatric surgery populations. There is limited research in bariatric surgery and micronutrient genetic variants.<h4>Conclusion</h4>Genotype-based PN is becoming feasible to provide an effective treatment of micronutrient deficiencies associated with bariatric surgery. The role of genomic technology in micronutrient recommendations needs further investigation.

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

Non-random chromosomal translocations involving the putative transcription factor Mixed Lineage Leukemia Translocated to 10 (MLLT10, also known as AF10) are commonly observed in both acute myeloid and lymphoid leukemias and are indicative of a poor prognosis. Despite the well-described actions of oncogenic MLLT10 fusion proteins, the role of wild-type MLLT10 in hematopoiesis is not well characterized. The protein structure and several interacting partners have been described and provide indications as to the potential functions of MLLT10. This review examines these aspects of MLLT10, contextualizing its function in benign and malignant hematopoiesis.

Huntington disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the HTT gene. Various HD animal models have been generated to mimic the motor, cognitive and neuropsychiatric disturbances that affect HD patients. Reproducing disease phenotypes within these models is essential to identify reliable readouts for therapy studies. We validated behavioral phenotypes shown earlier by other research groups in the BACHD rat model, using both previously applied and novel tests for motor, cognitive and anxiety-like behaviors. We first confirmed known BACHD rats' phenotypes in rotarod, open field (OF) and elevated plus maze (EPM) tests. We then assessed the reproducibility of key phenotypes in the model using new tests: cliff hanging, passive avoidance (PA), Morris water maze (MWM), light dark box and light spot tests. We confirmed impaired motor coordination in the rotarod test and reduced activity in the OF. In line with earlier results in BACHD rats using different tests, we showed impaired reversal learning in MWM and decreased anxiety-like behavior with the light spot test supporting the validity of BACHD rats as a model of HD. Results in the EPM, light dark box, cliff hanging and PA tests did not confirm earlier findings. This may depend on phenotype inconsistencies or rather be related to differences in environmental variables, test typology, experimental settings, animal age and chosen behavioral parameters.

Quantum Dot^®s (QDot^®s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot^®s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot^®s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot^®s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot^®s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog's stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.

Proper immune system function hinders cancer development, but little is known about whether genetic variants linked to cancer risk alter immune cells. Here, we report 57 cancer risk loci associated with differences in immune and/or stromal cell contents in the corresponding tissue. Predicted target genes show expression and regulatory associations with immune features. Polygenic risk scores also reveal associations with immune and/or stromal cell contents, and breast cancer scores show consistent results in normal and tumor tissue. SH2B3 links peripheral alterations of several immune cell types to the risk of this malignancy. Pleiotropic SH2B3 variants are associated with breast cancer risk in BRCA1/2 mutation carriers. A retrospective case-cohort study indicates a positive association between blood counts of basophils, leukocytes, and monocytes and age at breast cancer diagnosis. These findings broaden our knowledge of the role of the immune system in cancer and highlight promising prevention strategies for individuals at high risk.

Advances in the treatment of Ewing's sarcoma (EWS) are desperately needed, particularly in the case of metastatic disease. A deeper understanding of collateral sensitivity, where the evolution of therapeutic resistance to one drug aligns with sensitivity to another drug, may improve our ability to effectively target this disease. For the first time in a solid tumor, we produced a temporal collateral sensitivity map that demonstrates the evolution of collateral sensitivity and resistance in EWS. We found that the evolution of collateral resistance was predictable with some drugs but had significant variation in response to other drugs. Using this map of temporal collateral sensitivity in EWS, we can see that the path toward collateral sensitivity is not always repeatable, nor is there always a clear trajectory toward resistance or sensitivity. Identifying transcriptomic changes that accompany these states of transient collateral sensitivity could improve treatment planning for patients with EWS.

Ferroptosis is a type of cell death related to cancer; however, the characteristics of ferroptosis in cancers are still uncertain. Based on the data in The Cancer Genome Atlas, we found that most ferroptosis regulator genes (FRGs) were differentially expressed in tumors, somatic copy number alterations (SCNA) and DNA methylation contributed to their aberrant expression. We established the ferroptosis potential index (FPI) to reveal the functional roles of ferroptosis and noticed that the FPI was higher in tumors than in normal tissues in most cancers and was associated with subtypes and clinical features. The FPI was negatively correlated with several metabolic pathways but positively associated with several important metastasis-related pathways and immune-related pathways. High FPI predicted poor prognosis in several tumors, whereas FPI and FRGs impacted drug sensitivity. Our study presents a systematic analysis of ferroptosis and its regulatory genes and highlights the potential of ferroptosis-based cancer therapy.

<h4>Background</h4> Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates. As such, there is a pressing need to develop more effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use a multiomics approach to interrogate a collection of LGSOC patient-derived cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. <h4>Methods</h4> Fourteen LGSOC cell lines were interrogated using whole exome sequencing, RNA sequencing, and mass spectrometry-based proteomics. Somatic mutation, copy-number aberrations, gene and protein expression were analyzed and integrated using different computational approaches. LGSOC cell line data was compared to publicly available LGSOC tumor data (AACR GENIE cohort), and also used for predictive biomarker identification of MEK inhibitor (MEKi) efficacy. Protein interaction databases were evaluated to identify novel therapeutic targets. <h4>Results</h4> KRAS mutations were exclusively found in MEKi-sensitive and NRAS mutations mostly in MEKi-resistant cell lines. Analysis of COSMIC mutational signatures revealed distinct patterns of nucleotide substitution mutations in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes (chromosome 9p21) were much more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. For in-vitro MEKi efficacy prediction, proteomic data provided better discrimination than gene expression data. Condensin, MCM, and RFC protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. <h4>Conclusions</h4> Our LGSOC cell lines are representative models of the most common molecular aberrations found in LGSOC tumors. This study highlights the importance of using proteomic data in multiomics assessment of drug prediction and identification of potential therapeutic targets. CDKN2A/B and MTAP deficiency offer an opportunity to find synthetically lethal candidates for novel treatments. Multiomics approaches are crucial to improving our understanding of the molecular aberrations in LGSOC, establishing effective drug prediction programs and identifying novel therapeutic targets in LGSOC.

<h4> A bstract </h4> Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless – in some cases – reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV. <h4> I mportance </h4> The bifunctional nsp14 subunit of the coronavirus replicase contains 3’-to-5’ exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

In January 2020, we identified two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in a familial cluster with one person coming from Wuhan, China. The complete genome sequences of two SARS-CoV-2 strains isolated from these patients were identical and 99.98% similar to strains isolated in Wuhan. This is genetically suggestive of human-to-human transmission of SARS-CoV-2 and indicates Wuhan as the most plausible origin of the early outbreak in Vietnam. The younger patient had a mild upper respiratory illness and a brief viral shedding, whereas the elderly with multi-morbidity had pneumonia, prolonged viral shedding, and residual lung damage. The evidence of nonsynonymous substitutions in the ORF1ab region of the viral sequence warrants further studies.

Ambient air, occupational settings, and the use and distribution of consumer products all serve as conduits for toxicant exposure through inhalation. While the pulmonary system remains a primary target following inhalation exposure, cardiovascular implications are exceptionally culpable for increased morbidity and mortality. The epidemiological evidence for cardiovascular dysfunction resulting from acute or chronic inhalation exposure to particulate matter has been well documented, but the mechanisms driving the resulting disturbances remain elusive. In the current review, we aim to summarize the cellular and molecular mechanisms that are directly linked to cardiovascular health following exposure to a variety of inhaled toxicants. The purpose of this review is to provide a comprehensive overview of the biochemical changes in the cardiovascular system following particle inhalation exposure and to highlight potential biomarkers that exist across multiple exposure paradigms. We attempt to integrate these molecular signatures in an effort to provide direction for future investigations. This review also characterizes how molecular responses are modified in at-risk populations, specifically the impact of environmental exposure during critical windows of development. Maternal exposure to particulate matter during gestation can lead to fetal epigenetic reprogramming, resulting in long-term deficits to the cardiovascular system. In both direct and indirect (gestational) exposures, connecting the biochemical mechanisms with functional deficits outlines pathways that can be targeted for future therapeutic intervention. Ultimately, future investigations integrating "omics"-based approaches will better elucidate the mechanisms that are altered by xenobiotic inhalation exposure, identify biomarkers, and guide in clinical decision making.

The leading cause of death in Pulmonary Arterial Hypertension (PAH) is right ventricular (RV) failure. The tumor suppressor p53 has been associated with left ventricular hypertrophy (LVH) and remodeling but its role in RV hypertrophy (RVH) is unclear. The purpose of this study was to determine whether pharmacological activation of p53 by Quinacrine affects RV remodeling and function in the pulmonary artery banding (PAB) model of compensated RVH in mice. The effects of p53 activation on cellular functions were studied in isolated cardiomyocytes, cardiac fibroblasts and endothelial cells (ECs). The expression of p53 was examined both on human RV tissues from patients with compensated and decompensated RVH and in mouse RV tissues early and late after the PAB. As compared to control human RVs, there was no change in p53 expression in compensated RVH, while a marked upregulation was found in decompensated RVH. Similarly, in comparison to SHAM-operated mice, unaltered RV p53 expression 7 days after PAB, was markedly induced 21 days after the PAB. Quinacrine induced p53 accumulation did not further deteriorate RV function at day 7 after PAB. Quinacrine administration did not increase EC death, neither diminished EC number and capillary density in RV tissues. No major impact on the expression of markers of sarcomere organization, fatty acid and mitochondrial metabolism and respiration was noted in Quinacrine-treated PAB mice. p53 accumulation modulated the expression of Heme Oxygenase 1 (HO-1) and Glucose Transporter (Glut1) in mouse RVs and in adult cardiomyocytes. We conclude that early p53 activation in PAB-induced RVH does not cause substantial detrimental effects on right ventricular remodeling and function.

Macroautophagy ("autophagy") is the main lysosomal catabolic process that becomes activated under nutrient-depleted conditions, like amino acid (AA) starvation. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-conserved negative regulator of autophagy. While leucine (Leu) is a critical mTORC1 regulator under AA-starved conditions, how Leu regulates autophagy is poorly understood. Here, we describe that in most cell types, including neurons, Leu negatively regulates autophagosome biogenesis via its metabolite, acetyl-coenzyme A (AcCoA). AcCoA inhibits autophagy by enhancing EP300-dependent acetylation of the mTORC1 component raptor, with consequent activation of mTORC1. Interestingly, in Leu deprivation conditions, the dominant effects on autophagy are mediated by decreased raptor acetylation causing mTORC1 inhibition, rather than by altered acetylation of other autophagy regulators. Thus, in most cell types we examined, Leu regulates autophagy via the impact of its metabolite AcCoA on mTORC1, suggesting that AcCoA and EP300 play pivotal roles in cell anabolism and catabolism.

Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1^-/- mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.

Carrot (Daucus carota L.) is a biennial plant requiring vernalization to induce flowering, but long days can promote its premature bolting and flowering. The basic genetic network controlling the flowering time has been constructed for carrot, but there is limited information on the molecular mechanisms underlying the photoperiodic flowering response. The published carrot genome could provide an effective tool for systematically retrieving the key integrator genes of GIGANTEA (GI), CONSTANS-LIKE (COL), FLOWERING LOCUS T (FT), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) homologues in the photoperiod pathway. In this study, the bolting time of wild species "Songzi" (Ws) could be regulated by different photoperiods, but the orange cultivar "Amsterdam forcing" (Af) displayed no bolting phenomenon. According to the carrot genome and previous de novo transcriptome, 1 DcGI, 15 DcCOLs, 2 DcFTs, and 3 DcSOC1s were identified in the photoperiod pathway. The circadian rhythm peaks of DcGI, DcCOL2, DcCOL5a, and DcCOL13b could be delayed under long days (LDs). The peak value of DcCOL2 in Af (12.9) was significantly higher than that in Ws (6.8) under short day (SD) conditions, and was reduced under LD conditions (5.0). The peak values of DcCOL5a in Ws were constantly higher than those in Af under the photoperiod treatments. The expression levels of DcFT1 in Ws (463.0) were significantly upregulated under LD conditions compared with those in Af (1.4). These responses of DcCOL2, DcCOL5a, and DcFT1 might be related to the different bolting responses of Ws and Af. This study could provide valuable insights into understanding the key integrator genes in the carrot photoperiod pathway.

The stromal compartment of the tumor microenvironment consists of a heterogeneous set of tissue-resident and tumor-infiltrating cells, which are profoundly moulded by cancer cells. An outstanding question is to what extent this heterogeneity is similar between cancers affecting different organs. Here, we profile 233,591 single cells from patients with lung, colorectal, ovary and breast cancer (n = 36) and construct a pan-cancer blueprint of stromal cell heterogeneity using different single-cell RNA and protein-based technologies. We identify 68 stromal cell populations, of which 46 are shared between cancer types and 22 are unique. We also characterise each population phenotypically by highlighting its marker genes, transcription factors, metabolic activities and tissue-specific expression differences. Resident cell types are characterised by substantial tissue specificity, while tumor-infiltrating cell types are largely shared across cancer types. Finally, by applying the blueprint to melanoma tumors treated with checkpoint immunotherapy and identifying a naïve CD4⁺ T-cell phenotype predictive of response to checkpoint immunotherapy, we illustrate how it can serve as a guide to interpret scRNA-seq data. In conclusion, by providing a comprehensive blueprint through an interactive web server, we generate the first panoramic view on the shared complexity of stromal cells in different cancers.

Protein-protein interactions (PPIs) are important biochemical processes that represent a major challenge in modern biology. Current approaches, which include high-throughput screening and computer aided ligand design, have limitations regarding the identification of hit matter. This current investigation focuses on computational study for protein-protein docking of hypoxia inducible factor-1α (HIF-1α), a tumor inducible factor, and Raf-1 kinase inhibitory protein (RKIP), a tumor metastasis suppressor. These are individually crystallized structures of interacting proteins, which interact to generate a conformational space. HIF activity in pancreatic tumors is determined by hypoxia and HIF-1α subunit availability. RKIP can be used as a prognostic indicator in a number of tumors. The interaction of RKIP with HIF-1α protects against pancreatic cancer (PC) metastasis by inhibiting its hypoxia function. We have explored the binding affinity between both the proteins with the HADDOCK (high ambiguity driven protein-protein docking) server, which determined that 158 structures in 11 clusters represent 79.0% of water-refined models. Of the best 10 clusters, the structures of cluster 2 were found to be better, as they had the lowest Z-score. Further supporting HIF-1α-RKIP interaction, pulldown assay has shown dissociation of RKIP from HIF-1α after CoCl2 treatment in both PC cell lines.

Amyotrophic lateral sclerosis is a neurodegenerative disease of the upper and lower motor neurons resulting in death from neuromuscular respiratory failure, typically within two to five years of first symptoms. Several rare disruptive gene variants have been associated with ALS and are responsible for about 15% of all cases. Although our knowledge of the genetic landscape of this disease is improving, it remains limited. Machine learning models trained on the available protein-protein interaction and phenotype-genotype association data can use our current knowledge of the disease genetics for the prediction of novel candidate genes. Here, we describe a knowledge-based machine learning method for this purpose. We trained our model on protein-protein interaction data from IntAct, gene function annotation from Gene Ontology, and known disease-gene associations from DisGeNet. Using several sets of known ALS genes from public databases and a manual review as input, we generated a list of new candidate genes for each input set. We investigated the relevance of the predicted genes in ALS by using the available summary statistics from the largest ALS genome-wide association study and by performing functional and phenotype enrichment analysis. The predicted sets were enriched for genes associated with other neurodegenerative diseases known to overlap with ALS genetically and phenotypically, as well as for biological processes associated with the disease. Moreover, using ALS genes from ClinVar and our manual review as input, the predicted sets were enriched for ALS-associated genes (ClinVar <i>p</i> = 0.038 and manual review <i>p</i> = 0.060) when used for gene prioritisation in a genome-wide association study.

Improving the therapeutic efficacy of conventional anticancer drugs represents the best hope for cancer treatment. However, the shortage of druggable targets and the increasing development of anticancer drug resistance remain significant problems. Recently, membrane transport proteins have emerged as novel therapeutic targets for cancer treatment. These proteins are essential for a plethora of cell functions ranging from cell homeostasis to clinical drug toxicity. Furthermore, their association with carcinogenesis and chemoresistance has opened new vistas for pharmacology-based cancer research. This review provides a comprehensive update of our current knowledge on the functional expression profile of membrane transport proteins in cancer and chemoresistant tumours that may form the basis for new cancer treatment strategies.

Endometriosis is characterized by ectopic endometrial tissue implantation, mostly within the peritoneum, and affects women in their reproductive age. Studies have been done to clarify its etiology, but the precise molecular mechanisms and pathophysiology remain unclear. We downloaded genome-wide mRNA expression and clinicopathological data of endometriosis patients and controls from NCBI's Gene Expression Omnibus, after a systematic search of multiple independent studies comprising 156 endometriosis patients and 118 controls to identify causative genes, risk factors, and potential diagnostic/therapeutic biomarkers. Comprehensive gene expression meta-analysis, pathway analysis, and gene ontology analysis was done using a bioinformatics-based approach. We identified 1590 unique differentially expressed genes (129 upregulated and 1461 downregulated) mapped by IPA as biologically relevant. The top upregulated genes were <i>FOS</i>, <i>EGR1</i>, <i>ZFP36</i>, <i>JUNB</i>, <i>APOD</i>, <i>CST1</i>, <i>GPX3</i>, and <i>PER1</i>, and the top downregulated ones were <i>DIO2</i>, <i>CPM</i>, <i>OLFM4</i>, <i>PALLD</i>, <i>BAG5</i>, <i>TOP2A</i>, <i>PKP4</i>, <i>CDC20B</i>, and <i>SNTN</i>. The most perturbed canonical pathways were mitotic roles of Polo-like kinase, role of Checkpoint kinase proteins in cell cycle checkpoint control, and ATM signaling. Protein-protein interaction analysis showed a strong network association among FOS, EGR1, ZFP36, and JUNB. These findings provide a thorough understanding of the molecular mechanism of endometriosis, identified biomarkers, and represent a step towards the future development of novel diagnostic and therapeutic options.

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients' survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.

Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein-protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose-muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues.

The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (<i>n</i> = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 <i>v</i>/<i>v</i>) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.

<h4>Introduction</h4>To investigate effects of Sitagliptin on the enhancement of beta-cell function, reducing insulin resistance, serum glucagon like peptide-1 (GLP-1) concentrations and blood glucose in patients with type 2 diabetes mellitus (T2D) and suggest one of the underlying mechanisms on beta-cell function and insulin resistance.<h4>Patients and methods</h4>This was a cross-sectional and observational study in comparison to the control group. A study population of 44 newly diagnosed patients with T2D treated with Sitagliptin with a dose of 100 mg/day for 3 months was analyzed to compare 52 healthy participants. Indices for beta-cell function, peripheral insulin sensitivity, and insulin resistance were calculated with homeostasis model assessment 2 (HOMA2) calculator and compared. Serum GLP-1 concentrations were analyzed, and regression analysis was conducted to find the correlations between GLP-1 and beta-cell function and insulin resistance.<h4>Results</h4>Newly diagnosed patients with T2D witnessed a significant reduction in beta-cell function, serum GLP-1 concentrations at the time of diagnosis. After treatment with Sitagliptin 100 mg/day, they achieved significant improvements in beta-cell function, peripheral insulin sensitivity and insulin resistance. Serum GLP-1 concentrations were increased significantly to those levels in the control group and correlated with peripheral insulin sensitivity and insulin resistance in patients whose beta-cell functions improved.<h4>Conclusion</h4>Sitagliptin improved beta-cell function, insulin resistance and blood glucose in newly diagnosed patients with T2D. Meanwhile, Sitagliptin ameliorated serum GLP-1 concentrations, which contributed to the enhancement of beta-cell.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an unprecedented worldwide health problem that requires concerted and global approaches to stop the coronavirus 2019 (COVID-19) pandemic. Although SARS-CoV-2 primarily targets lung epithelium cells, there is growing evidence that the intestinal epithelium is also infected. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of the SARS-CoV-2 life cycle in human intestinal epithelial cells (hIECs). Our results demonstrate that hIECs fully support SARS-CoV-2 infection, replication, and production of infectious de novo virus particles. We found that viral infection elicits an extremely robust intrinsic immune response where interferon-mediated responses are efficient at controlling SARS-CoV-2 replication and de novo virus production. Taken together, our data demonstrate that hIECs are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing to increasing patient viremia and fueling an exacerbated cytokine response.

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold changes and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compared well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as fluorescence-activated cell sorting (FACS)-purified cells from the tunicate <i>Ciona</i>. Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

Cytokinins (CKs) are a diverse group of evolutionarily significant growth-regulating molecules. While the CK biosynthesis and signal transduction pathways are the most well-understood in plant systems, these molecules have been identified in all kingdoms of life. This review follows the recent discovery of an expanded CK profile in the social amoeba, <i>Dictyostelium discoideum</i>. A comprehensive review on the present knowledge of CK biosynthesis, signal transduction, and CK-small molecule interactions within members of Dictyostelia will be summarized. In doing so, the utility of social amoebae will be highlighted as a model system for studying the evolution of these hormone-like signaling agents, which will set the stage for future research in this area.

Corrosion-control coatings which can enhance bone formation and be completely replaced by bone are attractive for biodegradable Mg alloys. Carbonate apatite (CAp) and hydroxyapatite (HAp) coatings were formed on Mg-4 wt% Y-3 wt% rare earth (WE43) alloy as a corrosion-control and bioabsorbable coating in the coating solution with various concentrations of NaHCO₃. The incorporation of carbonate group in apatite structure was examined using X-ray diffraction and Fourier transform infrared spectroscopy. Rat osteoclast precursor and MC3T3-E1 osteoblast cells were cultured on the CAp- and HAp-coated WE43 to examine the osteoclastic resorption and the alkaline phosphatase (ALP) activity, respectively. Mg ions in the used medium were quantified to examine the corrosion-control ability. The NaHCO₃ addition in the solution resulted in the formation of B-type CAp in which the phosphate group of apatite structure was substituted with the carbonate group. The osteoclastic resorption was observed only for the CAp coatings as the cracking of the coatings and the corrosion of substrate WE43 strongly localized under osteoclast cell bodies. The CAp and HAp coatings significantly enhanced the ALP activity of osteoblasts. The CAp-coated WE43 specimens showed 1/5 smaller amount of Mg ion release than the uncoated WE43 on the first day of culturing osteoblasts. For the subsequent 22 days, the Mg ion release was reduced to 1/2 by the CAp coatings. In the presence of osteoclasts, the CAp coatings showed slightly lower corrosion protectiveness than the HAp coating. It was demonstrated that the CAp coatings can be a bioabsorbable and corrosion-control coating for biodegradable Mg alloys.

<h4>ABSTRACT</h4> <h4>BACKGROUND</h4> Epidemiological studies indicate that as many as 20% of individuals who test positive for COVID-19 develop severe symptoms that can require hospitalization. These symptoms include low platelet count, severe hypoxia, increased inflammatory cytokines and reduced glomerular filtration rate. Additionally, severe COVID-19 is associated with several chronic co-morbidities, including cardiovascular disease, hypertension and type 2 diabetes mellitus. The identification of genetic risk factors that impact differential host responses to SARS-CoV-2, resulting in the development of severe COVID-19, is important in gaining greater understanding into the biological mechanisms underpinning life-threatening responses to the virus. These insights could be used in the identification of high-risk individuals and for the development of treatment strategies for these patients. <h4>METHODS</h4> As of June 6, 2020, there were 976 patients who tested positive for COVID-19 and were hospitalized, indicating they had a severe response to SARS-CoV-2. To overcome the limited number of patients with a mild form of COVID-19, we used similar control criteria to our previous study looking at shared genetic risk factors between severe COVID-19 and sepsis, selecting controls who had not developed sepsis despite having maximum co-morbidity risk and exposure to sepsis-causing pathogens. <h4>RESULTS</h4> Using a combinatorial (high-order epistasis) analysis approach, we identified 68 protein-coding genes that were highly associated with severe COVID-19. At the time of analysis, nine of these genes have been linked to differential response to viral pathogens including SARS-CoV-2. We also found many novel targets that are involved in key biological pathways associated with the development of severe COVID-19, including production of pro-inflammatory cytokines, endothelial cell dysfunction, lipid droplets, neurodegeneration and viral susceptibility factors. <h4>CONCLUSION</h4> The variants we found in genes relating to immune response pathways and cytokine production cascades, were in equal proportions across all severe COVID-19 patients, regardless of their co-morbidities. This suggests that such variants are not associated with any specific co-morbidity, but are common amongst patients who develop severe COVID-19. This is consistent with being able to find and validate severe disease biomarker signatures when larger patient datasets become available. Several of the genes identified relate to lipid programming, beta-catenin and protein kinase C signalling. These processes converge in a central pathway involved in plasma membrane repair, clotting and wound healing. This pathway is largely driven by Ca 2+ activation, which is a known serum biomarker associated with severe COVID-19 and ARDS. This suggests that aberrant calcium ion signalling may be responsible for driving severe COVID-19 responses in patients with variants in genes that regulate the expression and activity of this ion. We intend to perform further analyses to confirm this hypothesis. Among the 68 severe COVID-19 risk-associated genes, we found several druggable protein targets and pathways. Nine are targeted by drugs that have reached at least Phase I clinical trials, and a further eight have active chemical starting points for novel drug development. Several of these targets were particularly enriched in specific co-morbidities, providing insights into shared pathological mechanisms underlying both the development of severe COVID-19, ARDS and these predisposing co-morbidities. We can use these insights to identify patients who are at greatest risk of contracting severe COVID-19 and develop targeted therapeutic strategies for them, with the aim of improving disease burden and survival rates.

<h4>Background: </h4> Magnetospirillum gryphiswaldense (MSR-1), a type of magnetotactic bacteria are capable of forming magnetosomes, which are nanometer-sized particles and membrane-enclosed organelles containing crystals of magnetite (Fe 3 O 4 ). For the synthesis of magnetosomes, bacteria require a significant amount of iron. Iron is an essential element for most life on earth; however, the elevation of iron level leads to hemochromatosis.This research aimed to investigate whether MSR-1 has an impact on the alteration of iron levels in mammalian cells in vivo. Female BALB/c were received intraperitonealinjections of iron-dextran (100mg/kg b.w.) 2 times per week for four consecutive weeks, and the iron was allowed to equilibrate for 15 days. The MSR-1 (10 9 in 200 µl PBS)were injected into healthy (C+MSR-1) and iron overloaded (IO+MSR-1) mice. The control healthy (C+ no MSR-1) and control iron overloaded (IO+ no MSR-1) mice received PBS. After the administration of bacteria, the viable bacterial numbers were evaluated in mice. Iron levels in serum and liver, as well as urinary iron exertion, were detected. <h4>Results: </h4> Bacteria were not detected in the liver, spleen, and lymph nodes of mice after 72 hours of post-injection. Serum iron and total liver iron levels in C+MSR-1 were reduced compare to C-MSR-1 (by 42.1% and 34.1%), respectively. Moreover, similar patterns were observed in IO+MSR-1 compare to IO+ no MSR-1, 42.1% iron reduction in serum, and 32.1% in the liver after 24h of post-injection. The urinary iron exertion was dramatically increased (by 3-fold) compared to groups with no MSR-1. <h4>Conclusion: </h4> Our results suggest that MSR-1 can reduce the iron in iron-overloaded mice and, therefore, may inhibit the organ damage caused by excess iron. Our research on optimizing the biological magnetic system is continuing.