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Endometriosis is widely associated with infertility in women of childbearing age, for which there have been no effective treatments. Recent studies suggest that the dysregulation of RNAs contributes to the pathogenesis of endometriosis, so we conduct the case-control genetic analysis to characterize the expression and interaction of different subtypes of RNAs in infertile women with endometriosis. The ectopic and eutopic endometrium of patients undergoing infertility treatment were collected and subjected to high throughput sequencing, and bioinformatics analysis was conducted to construct the competing endogenous RNA (ceRNA) network. As a result, the RNA interactive network was constructed in endometriosis, and a set of mRNAs such as cyclin-dependent kinase 1 (CDK1) and proliferating cell nuclear antigen (PCNA) along with their corresponding miRNAs and lncRNAs were found to promote the growth and death of endometrial stromal cells, which was essential for the pathogenesis of endometriosis. These data suggest that RNA crosstalk is a crucial segment in the development of endometriosis, where CDK1 and PCNA may serve as emerging targets for the treatment of endometriosis-related infertility in women of childbearing age.

Long non-coding RNA (lncRNA) is known to be involved in a variety of diseases. However, the role of Gm4419 in hepatic ischemia-reperfusion (I/R) injury remains unknown. To study this, we first established a rat model of hepatic I/R, and a BRL-3A cell model of hypoxia-reoxygenation (H/R) for in vivo and in vitro studies. Staining with hematoxylin and eosin and hepatic injury scores were used to evaluate the degree of hepatic I/R injury. Cell apoptosis was assessed via staining with Edu, and with annexin V-FITC-propidium iodide assays. The interactions between Gm4419 and miR-455, as well as miR-455 and SOX6 were evaluated via luciferase reporter activity assays and RNA immunoprecipitation assays. In vivo, we found that Gm4419 was up-regulated in the rats subjected to I/R. Moreover, knockdown of Gm4419 alleviated the I/R-induced liver damage in the rats. In vitro, knockdown of Gm4419 alleviated H/R-induced apoptosis in BRL-3A cells. Interestingly, we found that miR-455 is a target of Gm4419, and Gm4419 regulates the expression of miR-455 via sponging. Furthermore, SOX6 was proven to be the target of miR-455. Finally, rescue experiments confirmed that knockdown of Gm4419 inhibits apoptosis by regulating miR-455 and SOX6 in H/R-treated BRL-3A cells. Therefore, our findings show that the lncRNA Gm4419 accelerates hepatic I/R injury by targeting the miR-455-SOX6 axis, which suggests a novel therapeutic target for hepatic I/R injury.

Observational epidemiological studies indicate that endometriosis and migraine co-occur within individuals more than expected by chance. However, the aetiology and biological mechanisms underlying their comorbidity remain unknown. Here we examined the relationship between endometriosis and migraine using genome-wide association study (GWAS) data. Single nucleotide polymorphism (SNP) effect concordance analysis found a significant concordance of SNP risk effects across endometriosis and migraine GWAS. Linkage disequilibrium score regression analysis found a positive and highly significant genetic correlation (<i>r</i>_G = 0.38, <i>P</i> = 2.30 × 10^-25) between endometriosis and migraine. A meta-analysis of endometriosis and migraine GWAS data did not reveal novel genome-wide significant SNPs, and Mendelian randomisation analysis found no evidence for a causal relationship between the two traits. However, gene-based analyses identified two novel loci for migraine. Also, we found significant enrichment of genes nominally associated (<i>P</i>_gene < 0.05) with both traits (<i>P</i>_{binomial-test} = 9.83 × 10^-6). Combining gene-based p-values across endometriosis and migraine, three genes, two (<i>TRIM32 and SLC35G6)</i> of which are at novel loci, were genome-wide significant. Genes having <i>P</i>_gene < 0.1 for both endometriosis and migraine (<i>P</i>_{binomial-test} = 1.85 ×10^-°³) were significantly enriched for biological pathways, including interleukin-1 receptor binding, focal adhesion-PI3K-Akt-mTOR-signaling, MAPK and TNF-α signalling. Our findings further confirm the comorbidity of endometriosis and migraine and indicate a non-causal relationship between the two traits, with shared genetically-controlled biological mechanisms underlying the co-occurrence of the two disorders.

Ubiquitination, an important type of protein posttranslational modification (PTM), plays a crucial role in controlling substrate degradation and subsequently mediates the "quantity" and "quality" of various proteins, serving to ensure cell homeostasis and guarantee life activities. The regulation of ubiquitination is multifaceted and works not only at the transcriptional and posttranslational levels (phosphorylation, acetylation, methylation, etc.) but also at the protein level (activators or repressors). When regulatory mechanisms are aberrant, the altered biological processes may subsequently induce serious human diseases, especially various types of cancer. In tumorigenesis, the altered biological processes involve tumor metabolism, the immunological tumor microenvironment (TME), cancer stem cell (CSC) stemness and so on. With regard to tumor metabolism, the ubiquitination of some key proteins such as RagA, mTOR, PTEN, AKT, c-Myc and P53 significantly regulates the activity of the mTORC1, AMPK and PTEN-AKT signaling pathways. In addition, ubiquitination in the TLR, RLR and STING-dependent signaling pathways also modulates the TME. Moreover, the ubiquitination of core stem cell regulator triplets (Nanog, Oct4 and Sox2) and members of the Wnt and Hippo-YAP signaling pathways participates in the maintenance of CSC stemness. Based on the altered components, including the proteasome, E3 ligases, E1, E2 and deubiquitinases (DUBs), many molecular targeted drugs have been developed to combat cancer. Among them, small molecule inhibitors targeting the proteasome, such as bortezomib, carfilzomib, oprozomib and ixazomib, have achieved tangible success. In addition, MLN7243 and MLN4924 (targeting the E1 enzyme), Leucettamol A and CC0651 (targeting the E2 enzyme), nutlin and MI-219 (targeting the E3 enzyme), and compounds G5 and F6 (targeting DUB activity) have also shown potential in preclinical cancer treatment. In this review, we summarize the latest progress in understanding the substrates for ubiquitination and their special functions in tumor metabolism regulation, TME modulation and CSC stemness maintenance. Moreover, potential therapeutic targets for cancer are reviewed, as are the therapeutic effects of targeted drugs.

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat^-/- ), Gnpat/Hfe double-knockout (Gnpat^-/- Hfe^-/- or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpat^fl/fl ;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat^+/+ ) and Gnpat^-/- mice, between Hfe^-/- and DKO mice, or between Gnpat^fl/fl and Gnpat^fl/fl ;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat^-/- and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe^-/- mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe^-/- mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe^-/- mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.

The glycoproteins of hepatitis C virus, E1E2, are unlike any other viral fusion machinery yet described, and are the current focus of immunogen design in HCV vaccine development; thus, making E1E2 both scientifically and medically important. We used pre-existing, but fragmentary, structures to model a complete ectodomain of the major glycoprotein E2 from three strains of HCV. We then performed molecular dynamic simulations to explore the conformational landscape of E2, revealing a number of important features. Despite high sequence divergence, and subtle differences in the models, E2 from different strains behave similarly, possessing a stable core flanked by highly flexible regions, some of which perform essential functions such as receptor binding. Comparison with sequence data suggest that this consistent behaviour is conferred by a network of conserved residues that act as hinge and anchor points throughout E2. The variable regions (HVR-1, HVR-2 and VR-3) exhibit particularly high flexibility, and bioinformatic analysis suggests that HVR-1 is a putative intrinsically disordered protein region. Dynamic cross-correlation analyses demonstrate intramolecular communication and suggest that specific regions, such as HVR-1, can exert influence throughout E2. To support our computational approach we performed small-angle X-ray scattering with purified E2 ectodomain; this data was consistent with our MD experiments, suggesting a compact globular core with peripheral flexible regions. This work captures the dynamic behaviour of E2 and has direct relevance to the interaction of HCV with cell-surface receptors and neutralising antibodies.

<h4>Background</h4>Non-invasive monitoring of cerebral tissue oxygen saturation (rcSO₂) during transition is of growing interest. Different near-infrared spectroscopy (NIRS) techniques have been developed to measure rcSO₂. We compared rcSO₂ values during the immediate transition in preterm neonates measured with frequency-domain NIRS (FD-NIRS) with those measured with continuous-wave NIRS (CW-NIRS) devices in prospective observational studies.<h4>Methods</h4>We compared rcSO₂ values measured with an FD-NIRS device during the first 15 min after birth in neonates with a gestational age ≥ 30 weeks but < 37 weeks born at the Erasmus MC- Sophia Children's Hospital, Rotterdam, the Netherlands, with similar values measured with a CW-NIRS device in neonates born at the Medical University of Graz, Austria. Mixed models were used to adjust for repeated rcSO₂ measurements, with fixed effects for time (non-linear), device, respiratory support and the interaction of device and respiratory support with time. Additionally, parameters such as total haemoglobin concentration and oxygenated and deoxygenated haemoglobin concentrations measured by FD-NIRS were analysed.<h4>Results</h4>Thirty-eight FD-NIRS measurements were compared with 58 CW-NIRS measurements. The FD-NIRS rcSO₂ values were consistently higher than the CW-NIRS rcSO₂ values in the first 12 min, irrespective of respiratory support. After adjustment for respiratory support, the time-dependent trend in rcSO₂ differed significantly between techniques (p < 0.01).<h4>Conclusion</h4>As cerebral saturation measured with the FD-NIRS device differed significantly from that measured with the CW-NIRS device, differences in absolute values need to be interpreted with care. Although FD-NIRS devices have technical advantages over CW-NIRS devices, FD-NIRS devices may overestimate true cerebral oxygenation and their benefits might not outweigh the usability of the more clinically viable CW-NIRS devices.

<h4>Objective</h4>Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5⁺ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.<h4>Design</h4>Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60^Δ/ΔISC) and CD-like ileitis (TNF^ΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.<h4>Results</h4>In patients with CD and TNF^ΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased <i>Lgr5</i> expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNF^ΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNF^ΔARE mice-derived crypts to form organoids.<h4>Conclusion</h4>We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

Corpora lutea (CL) are transient endocrine glands supporting pregnancy by progesterone production. They develop at the site of ovulation from the remaining follicle, are highly metabolically active and undergo distinct, transformative processes during their lifetime. In contrast to other species, CL of lynxes do not regress at the end of cycle, but remain functionally active (persist) for years. Reactive oxygen species (ROS) and anti-oxidative enzymes are described to be important for the functionality of CL. We examined ten anti-oxidative enzymes in fresh and persistent CL of lynxes as well as in domestic cat CL of different luteal stages. The gene expression profiles, especially those of SOD1 and SOD2, showed some remarkable differences between CL stages during non-pregnant and pregnant cycles of domestic cats and between fresh and persistent CL of lynxes. Lynx gene expression profiles of SODs were confirmed by western blot analysis, immunohistochemistry and activity assays. SOD2 was characterized by a conspicuous high expression and enzyme activity exclusively in persistent CL. We suggest that SOD2 is required to detoxify potential elevated superoxide anion levels by producing H₂O₂ in the physiologically persistent CL. This product might also act as a signaling molecule, securing the CL from apoptosis and insuring long-term luteal cell survival.

As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

Telomeres are highly conserved tandem nucleotide repeats that include proximal double-stranded and distal single-stranded regions that in complex with shelterin proteins afford protection at chromosomal ends to maintain genomic integrity. Due to the inherent limitations of DNA replication and telomerase suppression in most somatic cells, telomeres undergo age-dependent incremental attrition. Short or dysfunctional telomeres are recognized as DNA double-stranded breaks, triggering cells to undergo replicative senescence. Telomere shortening, therefore, acts as a counting mechanism that drives replicative senescence by limiting the mitotic potential of cells. Telomere length, a complex hereditary trait, is associated with aging and age-related diseases. Epidemiological data, in general, support an association with varying magnitudes between constitutive telomere length and several disorders, including cancers. Telomere attrition is also influenced by oxidative damage and replicative stress caused by genetic, epigenetic, and environmental factors. Several single nucleotide polymorphisms at different loci, identified through genome-wide association studies, influence inter-individual variation in telomere length. In addition to genetic factors, environmental factors also influence telomere length during growth and development. Telomeres hold potential as biomarkers that reflect the genetic predisposition together with the impact of environmental conditions and as targets for anti-cancer therapies.

The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.

Ferroptosis has emerged as a new type of cell death in different pathological conditions, including neurological and kidney diseases and, especially, in different types of cancer. The hallmark of this regulated cell death is the presence of iron-driven lipid peroxidation; the activation of key genes related to this process such as glutathione peroxidase-4 (<i>gpx4</i>), acyl-CoA synthetase long-chain family member-4 (<i>acsl4</i>), carbonyl reductase [NADPH] 3 (<i>cbr3</i>), and prostaglandin peroxidase synthase-2 (<i>ptgs2</i>); and morphological changes including shrunken and electron-dense mitochondria. Iron overload in the liver has long been recognized as both a major trigger of liver damage in different diseases, and it is also associated with liver fibrosis. New evidence suggests that ferroptosis might be a novel type of non-apoptotic cell death in several liver diseases including non-alcoholic steatohepatitis (NASH), alcoholic liver disease (ALD), drug-induced liver injury (DILI), viral hepatitis, and hemochromatosis. The interaction between iron-related lipid peroxidation, cellular stress signals, and antioxidant systems plays a pivotal role in the development of this novel type of cell death. In addition, integrated responses from lipidic mediators together with free iron from iron-containing enzymes are essential to understanding this process. The presence of ferroptosis and the exact mechanisms leading to this non-apoptotic type of cell death in the liver remain scarcely elucidated. Recognizing ferroptosis as a novel type of cell death in the liver could lead to the understanding of the complex interaction between different types of cell death, their role in progression of liver fibrosis, the development of new biomarkers, as well as the use of modulators of ferroptosis, allowing improved theranostic approaches in the clinic.

<h4>Background and aims</h4>Neuroimmune guidance cues have been shown to play a role in atherosclerosis, but their exact role in human pathophysiology is largely unknown. In the current study, we investigated the role of a c.1769G > T variant in Netrin-1 in (premature) atherosclerosis.<h4>Methods</h4>To determine the effect of the genetic variation, purified Netrin-1, either wild type (wtNetrin-1) or the patient observed variation (mutNetrin-1), was used for migration, adhesion, endothelial barrier function and bindings assays. Expression of adhesion molecules and transcription proteins was analyzed by RT-PCR, Western blot or ELISA. To further delineate how mutNetrin-1 mediates its effect on cell migration, lenti-viral knockdown of UNC5B or DCC was used.<h4>Results</h4>Bindings assays revealed a decreased binding capacity of mutNetrin-1 to the receptors UNC5B, DCC and β3-integrin and an increased binding capacity to neogenin, heparin and heparan sulfate compared to wtNetrin-1. Exposure of endothelial cells to mutNetrin-1 resulted in enhanced monocyte adhesion and expression of IL-6, CCL2 and ICAM-1 compared to wtNetrin-1. In addition, mutNetrin-1 lacks the inhibitory effect on the NF-κB pathway that is observed for wtNetrin-1. Moreover, the presence of mutNetrin-1 diminished migration of macrophages and smooth muscle cells. Importantly, UNC5B or DCC specific knockdown showed that mutNetrin-1 is unable to act through DCC resulting in enhanced inhibition of migration.<h4>Conclusions</h4>Our data demonstrates that mutNetrin-1 fails to exert anti-inflammatory effects on endothelial cells and more strongly blocks macrophage migration compared to wtNetrin-1, suggesting that the carriers of this genetic molecular variant may well be at risk for premature atherosclerosis.

Being the center of the hypothalamus-pituitary-ovary (HPO) axis, the pituitary plays a key role in the onset of puberty. Recent studies show that circular RNAs (circRNAs) can perform as miRNA sponges to regulate development in animals. However, the function of pituitary-derived circRNAs in first estrus remains unclear in pigs. In this study, we performed a genome-wide identification and characterization of circRNAs using pituitaries from Landrace × Yorkshire crossbred pigs at three stages: pre-, in-, and post-puberty, to describe such pituitary-derived circRNAs in pigs. A total of 5148 circRNAs were found in the gilts' pituitaries, averaging 18 682 bp in genomic distance, which consisted of approximately 91% exonic, 6% intergenic, and 3% intronic circRNAs. Furthermore, 158 novel circRNAs were identified for the first time and classified as putative pituitary-specific circRNAs. Their expression levels during the onset of puberty, significantly exceeded those of the other circRNAs, and the parental genes of these putative pituitary-specific circRNAs were enriched in "ssc04917: prolactin signaling pathway," "ssc04080: neuroactive ligand-receptor interaction," and "ssc04728: dopaminergic synapse" pathways, all of which were consistent with pituitary functioning. Additionally, 17 differentially regulated circRNAs were found and investigated for their potential interaction with miRNAs, along with genes, by constructing a circRNA-targeted miRNA-gene network. Taken together, these results provide new insight into the circRNA-mediated timing of puberty in gilts at the pituitary level.

The development of the nanosized delivery systems with targeting navigation and efficient cargo release for cancer therapy has attracted great attention in recent years. Herein, a folic acid (FA) modified PEGylated polycaprolactone containing ditelluride linkage was synthesized through a facile coupling reaction. The hydrophobic doxorubicin (DOX) can be encapsulated into the polymeric micelles, and such nanoparticles (F-TeNP_DOX) exhibited redox-responsive drug release under abundant glutathione (GSH) condition due to the degradation of ditelluride bonds. In addition, flow cytometric analyses showed that the FA ligands on F-TeNP_DOX could facilitate their cellular uptake in 4T1 breast cancer cells. Therefore, F-TeNP_DOX led to the promoted drug accumulation and enhanced growth inhibition on 4T1 tumor <i>in vivo</i>. The obtained results suggest F-TeNP_DOX excellent potential as nanocarriers for anticancer drug delivery.

Interactions among genomic loci have often been overlooked in genome-wide association studies, revealing the combinatorial effects of variants on phenotype or disease manifestation. Unexplained genetic variance, interactions among causal genes of small effects, and biological pathways could be identified using a network biology approach. The main objective of this study was to determine the genome-wide epistatic variants affecting feed efficiency traits [feed conversion ratio (FCR) and residual feed intake (RFI)] based on weighted interaction SNP hub (WISH-R) method. Herein, we detected highly interconnected epistatic SNP modules, pathways, and potential biomarkers for the FCR and RFI in Duroc and Landrace purebreds considering the whole population, and separately for low and high feed efficient groups. Highly interacting SNP modules in Duroc (1,247 SNPs) and Landrace (1,215 SNPs) across the population and for low feed efficient (Duroc-80 SNPs, Landrace-146 SNPs) and high feed efficient group (Duroc-198 SNPs, Landrace-232 SNPs) for FCR and RFI were identified. Gene and pathway analyses identified <i>ABL1</i>, <i>MAP3K4</i>, <i>MAP3K5</i>, <i>SEMA6A</i>, <i>KITLG</i>, and <i>KAT2B</i> from chromosomes 1, 2, 5, and 13 underlying ErbB, Ras, Rap1, thyroid hormone, axon guidance pathways in Duroc. <i>GABBR2</i>, <i>GNA12,</i> and <i>PRKCG</i> genes from chromosomes 1, 3, and 6 pointed towards thyroid hormone, cGMP-PKG and cAMP pathways in Landrace. From Duroc low feed efficient group, the <i>TPK1</i> gene was found involved with thiamine metabolism, whereas <i>PARD6G</i>, <i>DLG2, CRB1</i> were involved with the hippo signaling pathway in high feed efficient group. <i>PLOD1</i> and <i>SETD7</i> genes were involved with lysine degradation in low feed efficient group in Landrace, while high feed efficient group pointed to genes underpinning valine, leucine, isoleucine degradation, and fatty acid elongation. Some SNPs and genes identified are known for their association with feed efficiency, others are novel and potentially provide new avenues for further research. Further validation of epistatic SNPs and genes identified here in a larger cohort would help to establish a framework for modelling epistatic variance in future methods of genomic prediction, increasing the accuracy of estimated genetic merit for FE and helping the pig breeding industry.

T-cell acute lymphoblastic leukemia/lymphoma is an aggressive hematological neoplasm whose classification is still based on immunophenotypic findings. Frontline treatment encompass high intensity combination chemotherapy with good overall survival; however, relapsing/refractory patients have very limited options. In the last years, the understanding of molecular physiopathology of this disease, lead to the identification of a subset of patients with peculiar genetic profile, namely "early T-cell precursors" lymphoblastic leukemia, characterized by dismal outcome and indication to frontline allogeneic bone marrow transplant. In general, the most common mutations occur in the NOTCH1/FBXW7 pathway (60% of adult patients), with a positive prognostic impact. Other pathogenic steps encompass transcriptional deregulation of oncogenes/oncosuppressors, cell cycle deregulation, kinase signaling (including IL7R-JAK-STAT pathway, PI3K/AKT/mTOR pathway, RAS/MAPK signaling pathway, ABL1 signaling pathway), epigenetic deregulation, ribosomal dysfunction, and altered expression of oncogenic miRNAs or long non-coding RNA. The insight in the genomic landscape of the disease paves the way to the use of novel targeted drugs that might improve the outcome, particularly in relapse/refractory patients. In this review, we analyse available literature on T-ALL pathogenesis, focusing on molecular aspects of clinical, prognostic, and therapeutic significance.

The present research reports a detailed in silico analysis of chromatin assembly factor-1 (CAF-1) family in human malaria parasite <i>Plasmodium falciparum</i>. Our analysis revealed five chromatin assembly factor-1 genes in <i>P. falciparum</i> (<i>Pf</i>CAF-1) and the <i>Pf</i>CAF-1 family was divided into two classes where, Class A belongs to the CAF-1 complex and others are kept in Class B. For comparative studies, orthologs of <i>Pf</i>CAF-1 family were identified across 53 eukaryotic species and evolutionary relationships were drawn for different CAF-1 subfamilies. The phylogenetic analysis revealed grouping of evolutionary-related species together, although, divergence was observed in branching pattern. A detailed analysis of domain composition highlighted species-specific features viz<i>.</i> species-specific KDDS repeats of 84 amino acids were identified in <i>Pf</i>CAF-1A whereas, members of CAF-1C/RbAp48 and RbAp46 subfamily exhibited least variation in size and domain composition. The qRT-PCR analysis revealed upregulation of <i>Pf</i>CAF-1 members in trophozoite or schizont stage. Furthermore, a comparative expression analysis of the available transcriptome and proteome data along with qRT-PCR analysis revealed mixed expression patterns (coordination as well as non-coordination between different studies). Protein-protein interaction network analyses of <i>Pf</i>CAF-1 family were carried out highlighting important complexes based on interologs. The <i>Pf</i>RbAp48 was found to be highly connected with a total of 108 PPIs followed by <i>Pf</i>RbAp46. The results unravel insights into the <i>Pf</i>CAF-1 family and identify unique features, thus opening new perspectives for further targeted developments to understand and combat malaria menace.

This experiment was conducted to examine the effect of 2 phosphorus (P) sources on broiler performance to day 14. The P bioavailability was estimated using bird performance and tibia ash measurements, whereas P digestibility, intestinal P transporter, kidney vitamin D-1α-hydroxylase, and vitamin D-24-hydroxylase mRNA abundances were also determined. Slope regression analysis was used to determine the bioavailability of dicalcium phosphate (Dical P) and nanocalcium phosphate (Nano P) with dietary available P (AvP) set to 0.20% P (control) using AvP from the major ingredients and Dical P. The experimental treatments were achieved by supplementation with either Dical P or Nano P to generate 0.24, 0.28, 0.32, and 0.36% AvP. A total of 648-day-old unsexed broiler chicks were divided into 72 birds per treatment (8 replicate cages of 9 birds). Slope regression analysis showed positive linear relationships between BW, feed intake (FI), tibia ash weight (TAW), and tibia ash percentage (TAP) with dietary Dical P and Nano P levels. Comparisons between regression slopes for Dical P and Nano P fed birds were not significantly different for BW, feed intake, tibia ash weight, and tibia ash percentage, indicating similar P bioavailability from Dical P and Nano P. There were interactions between P source and AvP for feed efficiency (FE) and apparent ileal P digestibility (AIPD). Dicalcium phosphate had greater FE than Nano P at 0.28% AvP and greater AIPD than Nano P at 0.24% AvP. The addition of AvP from Dical P and Nano P resulted in reduced sodium phosphate cotransporter mRNA abundance in the duodenum in a dose-dependent response. In the kidney, vitamin D-1α-hydroxylase mRNA abundance was greater at 0.36% Nano P compared with control, but there was no difference with Dical P. There was no difference in vitamin D-24-hydroxylase mRNA abundance between control and supplementation with Nano P or Dical P. In conclusion, Nano P and Dical P had the same bioavailability but had different effects on gene expression.

<h4>Introduction</h4>Esophageal varices develop as a consequence of portal hypertension (PHT) in patients with chronic liver disease. Hence, screening of all cirrhotic patients with upper gastrointestinal endoscopy to detect the presence of significant esophageal varices implies a number of unnecessary endoscopies and has its limitation where such facilities are not available, especially in the rural part of country.<h4>Method</h4>Patients with either sex, aged between 18 and 60 years with diagnosis of cirrhosis were studied. Detailed history, physical examination along with relevant investigations were recorded and upper gastrointestinal endoscopy was done within 2-3 days of investigation. Esophageal varices were graded as I-IV, using the Paquet grading system and patients were classified dichotomously either as having large esophageal varices (LEV) group A (Grade III-IV) and no varices group B (grade I-II).<h4>Result</h4>A total of 50 patients with cirrhosis of liver were recruited in the study. Among hematological markers, only low platelet count was significantly associated with the presence of LEV (<i>P</i> value <0.05). None of the biochemical markers were found to be significantly associated with LEV. All the ultrasonographic parameters, i.e. spleen size, splenic vein size, portal vein size, and the presence of portosystemic collaterals were found to be significantly associated with the presence of LEV (<i>P</i> value <0.05).<h4>Conclusion</h4>Though upper gastrointestinal endoscopy remains the gold standard for the diagnosis of esophageal varices in cirrhotic patients,those patients at high risk of having LEV can be screened by using clinical, hematological, biochemical, and radiological markers.

<h4>Background</h4>Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by the infiltration of fat in the liver cells. High levels of fat in the liver associated with increased risk of diseases like diabetes, high blood pressure and kidney disease, high cholesterol, metabolic syndrome.<h4>Aim and objective</h4>To determine the association between NAFLD and carotid intima media thickness (CIMT) among nondiabetic individuals.<h4>Materials and methods</h4>A total of 90 in-patients and outpatients (45 NAFL subjects and 45 patients with the normal liver as controls) with NAFL on abdomen ultrasound scan from Bangalore Baptist Hospital were included in our study. All the participants were subjected to a doppler study for carotid intima thickness and a blood sample (4 ml) was collected from all the subjects.<h4>Results</h4>In this study, there is an elevated level of BMI, weight, dyslipidemia in NAFLD compared with controls. The present study suggests that cardiovascular risk factors such as increased carotid intima-media thickness occur more frequently among NAFLD patients compared to healthy individuals.<h4>Conclusion</h4>We can conclude that NAFL has a significant association with higher cardiovascular risk in terms of carotid intima-media thickness, dyslipidemia, and hypoalphalipidemia. Lifestyle changes include weight loss, dietary changes, reduction of sedentary habits and physical exercise are recommended in the NAFL disease subjects with higher BMI and weight.

Immune-mediated inflammatory diseases are a group of diseases characterized by generalized inflammation that results from immune dysregulation, especially involving the mechanisms of acquired immunity. These diseases may be familial, showing that genetic factors play an important role in their development. Additionally, the occurrence of one disease makes a patient prone to other diseases. However, the coexistence of systemic lupus erythematosus (SLE) and psoriasis (Ps) is very rare due to their distinct genetic determinants and mechanisms of pathogenesis. Treatment is also challenging, as medications used to treat one condition exacerbate or even trigger the symptoms of the other. This paper presents the case of a Ps patient with a family history of autoimmune diseases, who developed systemic lupus erythematosus during puberty, as well as a discussion on the coexistence of SLE and Ps in developmental age based on available literature searching for PubMed database and American College of Rheumatology and European League Against Rheumatism abstracts particularly in this subject.

Betulinic acid is validated as a new self-assembly inducer for the formation of nanoparticles (NPs) in combination with different drugs. The target compounds are characterized by the presence of anticancer drugs acting on tubulin dynamics and of a linker that could be a carbon chain or a triazole-based one. Nanoparticles formed are characterized and their biological activity is evaluated.

Protein-protein interactions (PPIs) play an important role in numerous biological processes such as cell-cycle regulation and multiple diseases. The family of 14-3-3 proteins is an attractive target as they serve as binding partner to various proteins and are therefore capable of regulating their biological activities. Discovering small-molecule modulators, in particular stabilizers, of such complexes via traditional screening approaches is a challenging task. Herein, we pioneered the first application of dynamic combinatorial chemistry (DCC) to a PPI target, to find modulators of 14-3-3 proteins. Evaluation of the amplified hits from the DCC experiments for their binding affinity via surface plasmon resonance (SPR), revealed that the low-micromolar (<i>K</i> _D 15-16 μM) acylhydrazones are 14-3-3/synaptopodin PPI stabilizers. Thus, DCC appears to be ideally suited for the discovery of not only modulators but even the more elusive stabilizers of notoriously challenging PPIs.

Comprehensive understanding of aberrant splicing in gastric cancer is lacking. We RNA-sequenced 19 gastric tumor-normal pairs and identified 118 high-confidence tumor-associated (TA) alternative splicing events (ASEs) based on high-coverage sequencing and stringent filtering, and also identified 8 differentially expressed splicing factors (SFs). The TA ASEs occurred in genes primarily involved in cytoskeletal organization. We constructed a correlative network between TA ASE splicing ratios and SF expression, replicated it in independent gastric cancer data from The Cancer Genome Atlas and experimentally validated it by knockdown of the nodal SFs (<i>PTBP1</i>, <i>ESRP2</i> and <i>MBNL1</i>). Each SF knockdown drove splicing alterations in several corresponding TA ASEs and led to alterations in cellular migration consistent with the role of TA ASEs in cytoskeletal organization. We have therefore established a robust network of dysregulated splicing associated with tumor invasion in gastric cancer. Our work is a resource for identifying oncogenic splice forms, SFs and splicing-generated tumor antigens as biomarkers and therapeutic targets.

A series of 24 compounds were synthesized based on structure modification of the model methyl-3-(4-chlorophenyl)-3-hydroxy-2,2-dimethylpropanoate as potent HDACIs. Saponification and hydrazinolysis of the model ester afforded the corresponding acid and hydrazide, respectively. The model ester was transformed into the corresponding trichloroacetimidate or acetate by the reaction with trichloroacetonitrile and acetic anhydride, respectively. <i>N</i>-Alkyl-3-(4-chlorophenyl)-3-hydroxy-2,2-dimethylpropan-amides and methyl-2-[(3-(4-chlorophenyl)-3-hydroxy-2,2-dimethylpropanoyl)amino] alkanoates were obtained by the reaction of corresponding acid or hydrazide with amines and amino acid esters <i>via</i> DCC and azide coupling methods. Methyl-3-aryl-3-(4-chlorophenyl)-2,2-dimethylpropanoates were obtained in good yields and short reaction time from the corresponding trichloroacetimidate or acetate by the reaction with C-active nucleophiles in the presence of TMSOTf (0.1 eq.%) <i>via</i> C-C bond formation. The antiproliferative and apoptotic activity were further studied with molecular docking. The 48 post-treatments showed that out of 24 compounds, 12 compounds showed inhibitory actions on HCT-116 cells, we have calculated the inhibitory action (IC₅₀) of these compounds on HCT-116 and we have found that the IC₅₀ values were in between 0.12 mg mL^-1 to 0.81 mg mL^-1. The compounds (7a & 7g) showed highest inhibitory activity (0.12 mg mL^-1), whereas compound 7d showed the lowest inhibitory activity (0.81 mg mL^-1). We have also examined inhibitory action on normal and non-cancerous cells (HEK-293 cells) and confirmed that action of these compounds was specific to cancerous cells. The cancerous cells were also examined for nuclear disintegration through staining with DAPI, (4',6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain, and we have found that there was loss of DAPI staining in the compound treated cancerous cells. The compounds were found to potentially act through the HSP90 and TRAP1 mediated signaling pathway. Compounds 7a and 7g showed the highest selectivity to TRAP1 which explained its superior activity.

Irradiation, or chemoradiotherapy, is a curative treatment for oropharyngeal squamous cell carcinoma (OPSCC). Its invasiveness, however, can often negate its efficacy. Therefore, developing methods to predict which patients would benefit from irradiation is urgent. Promoter DNA hypermethylation was recently reported to correlate with favorable OPSCC prognosis. It is still unclear, however, whether there is an association between promoter DNA methylation and response to irradiation. In this study, we analyzed DNA methylation in the specimens from 40 OPSCC patients who had undergone irradiation, using the Infinium assay. Our results showed significant correlation between high levels of promoter DNA methylation and better response to treatment (P < 0.01). We used the 10 most differentially-methylated genes between responders and non-responders to develop a panel of predictive markers for efficacy. Our panel had high sensitivity, specificity and accuracy (92%, 93% and 93%, respectively). We conducted pyrosequencing to quantitatively validate the methylation levels of 8 of the 10 marker genes (ROBO1, ULK4P3, MYOD1, LBX1, CACNA1A, IRX4, DPYSL3 and ELAVL2) obtained by Infinium. The validation by pyrosequencing showed that these 8 genes had a high prediction performance for the training set of 40 specimens and for a validation set of 35 OPSCC specimens, showing 96% sensitivity, 89% specificity and 94% accuracy. Methylation of these markers correlated significantly with better progression-free and overall survival rates, regardless of human papillomavirus status. These results indicate that increased DNA methylation is associated with better responses to irradiation therapy and that DNA methylation can help establish efficacy prediction markers in OPSCC.

Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation, and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for liquid chromatography-mass spectrometry (LC-MS) analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylation state of kinases with unprecedented sensitivity. We also found that our kinobead/LC-MS protocol can measure changes in kinase complex composition and show how these changes can indicate kinase activity. We demonstrate the utility of our approach in specifying kinase signaling pathways that control the acute steroidogenic response in Leydig cells; this analysis establishes the first comprehensive framework for the post-translational control of steroid biosynthesis.

Molecular motor proteins form the basis of cellular dynamics. Recently, notable efforts have led to the creation of their DNA-based mimics, which can carry out complex nanoscale motion. However, such functional analogues have not yet been integrated or operated inside synthetic cells toward the goal of realizing artificial biological systems entirely from the bottom-up. In this Letter, we encapsulate and actuate DNA-assembled dynamic nanostructures inside cell-sized microfluidic compartments. These encapsulated DNA nanostructures not only exhibit structural reconfigurability owing to their pH-sensitive molecular switches upon external stimuli but also possess optical feedback enabled by the integrated plasmonic probes. In particular, we demonstrate the power of microfluidic compartmentalization for achieving on-chip plasmonic enantiomer separation and substrate filtration. Our work exemplifies that the two unique tools, droplet-based microfluidics and DNA technology, offering high precision on the microscale and nanoscale, respectively, can be brought together to greatly enrich the complexity and diversity of functional synthetic systems.

Due to side-effects and inefficiency of the drugs used in malaria treatment, finding alternative medicine with less side-effects has attracted much attention. In this regard, in the present study, nanocomposite synthesized and its effects on the metabolites of P. falciparum were investigated. Subsequent to synthesis of nanocomposites, characterization was carried out using nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC-MS), scanning electron microscopy, dynamic light scattering and Fourier-transform infrared tests. Solubility and drug release were measured and its toxicity on Vero cell was assessed using the MTT assay. The antiparasitic effect of the nanocomposite on the metabolites of P. falciparum was investigated by 1H NMR spectroscopy. Among synthesized nanocomposites, the average size of 239 nm showed suitable solubility in water as well as slow drug release. The MTT assay showed no toxicity for Vero cell lines. Concentrations of 2.5 μg mL-1 of nanocomposite eliminated 82.6% of the total parasites. The most effected metabolic cycles were glyoxylate and dicarboxylate metabolism. In this study, 1H NMR spectroscopy was used with untargeted metabolomics to study the effect of the nanocomposite on P. falciparum. Playing an essential role in understanding drug-target interactions and characterization of mechanism of action or resistance exhibited by novel antiprotozoal drugs, can be achieved by targeting metabolic using LC-MS.

Huntington's disease (HD) is caused by Huntingtin (Htt) gene mutation resulting in the loss of striatal GABAergic neurons and motor functional deficits. We report here an in vivo cell conversion technology to reprogram striatal astrocytes into GABAergic neurons in both R6/2 and YAC128 HD mouse models through AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors. We found that the astrocyte-to-neuron (AtN) conversion rate reached 80% in the striatum and >50% of the converted neurons were DARPP32⁺ medium spiny neurons. The striatal astrocyte-converted neurons showed action potentials and synaptic events, and projected their axons to the targeted globus pallidus and substantia nigra in a time-dependent manner. Behavioral analyses found that NeuroD1 and Dlx2-treated R6/2 mice showed a significant extension of life span and improvement of motor functions. This study demonstrates that in vivo AtN conversion may be a disease-modifying gene therapy to treat HD and other neurodegenerative disorders.

Micro(mi)RNA-based post-transcriptional regulatory mechanisms have been broadly implicated in the assembly and modulation of synaptic connections required to shape neural circuits, however, relatively few specific miRNAs have been identified that control synapse formation. Using a conditional transgenic toolkit for competitive inhibition of miRNA function in Drosophila, we performed an unbiased screen for novel regulators of synapse morphogenesis at the larval neuromuscular junction (NMJ). From a set of ten new validated regulators of NMJ growth, we discovered that miR-34 mutants display synaptic phenotypes and cell type-specific functions suggesting distinct downstream mechanisms in the presynaptic and postsynaptic compartments. A search for conserved downstream targets for miR-34 identified the junctional receptor CNTNAP4/Neurexin-IV (Nrx-IV) and the membrane cytoskeletal effector Adducin/Hu-li tai shao (Hts) as proteins whose synaptic expression is restricted by miR-34. Manipulation of miR-34, Nrx-IV or Hts-M function in motor neurons or muscle supports a model where presynaptic miR-34 inhibits Nrx-IV to influence active zone formation, whereas, postsynaptic miR-34 inhibits Hts to regulate the initiation of bouton formation from presynaptic terminals.

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.

Glycine N-myristoylation is an essential acylation modification modulating the functions, stability, and membrane association of diverse cytosolic proteins in human cells. Myristoyl-CoA is the 14-carbon acyl donor of the acyltransferase reaction. Acyl-CoAs of a chain length compatible with the binding site of the N-myristoyltransferase enzymes (NMT) are competitive inhibitors, and the mechanism protecting these enzymes from unwanted acyl-CoA species requires the acyl-CoA binding protein ACBD6. The acyl-CoA binding domain (ACB) and the ankyrin-repeat motifs (ANK) of ACBD6 can perform their functions independently. Interaction of ANK with human NMT2 was necessary and sufficient to provide protection. Fusion of the ANK module to the acyl-CoA binding protein ACBD1 was sufficient to confer the NMT-stimulatory property of ACBD6 to the chimera. The ACB domain is dispensable and sequestration of the competitor was not the basis for NMT2 protection. Acyl-CoAs bound to ACB modulate the function of the ANK module and act as positive effector of the allosteric activation of the enzyme. The functional relevance of homozygous mutations in ACBD6 gene, which have not been associated with a disease so far, is presented. Skin-derived fibroblasts of two unrelated individuals with neurodevelopmental disorder and carrying loss of function mutations in the ACBD6 gene were deficient in protein N-myristoylation. These cells were sensitive to substrate analog competing for myristoyl-CoA binding to NMT. These findings account for the requirement of an ANK-containing acyl-CoA binding protein in the cellular mechanism protecting the NMT enzymes and establish that in human cells, ACBD6 supports the N-myristoylation of proteins.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1⁺ neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.

Maintaining a healthy proteome throughout life is critical for proper somatic stem cell function, but the complexities of the stem cell response to increases in damaged or aggregated proteins remain unclear. Here we demonstrate that adult neural stem cells (NSCs) utilize aggresomes to recover from disrupted proteostasis and describe a novel function for the intermediate filament vimentin in proteostasis as a spatial coordinator of proteasomes to the aggresome. In the absence of vimentin, NSCs have a reduced capacity to exit quiescence, a time when NSCs are required to clear a wave of aggregated proteins, and demonstrate an early age-dependent decline in proliferation and neurogenesis. Taken together, these data reveal a significant role of vimentin and aggresomes in the regulation of proteostasis during quiescent NSC activation.

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novo-designed prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.

Genome-wide CRISPR screens enable systematic interrogation of gene function. However, guide RNA libraries are costly to synthesize, and their limited diversity compromises the sensitivity of CRISPR screens. Using the Streptococcus pyogenes CRISPR-Cas adaptation machinery, we developed CRISPR adaptation-mediated library manufacturing (CALM), which turns bacterial cells into "factories" for generating hundreds of thousands of crRNAs covering 95% of all targetable genomic sites. With an average gene targeted by more than 100 distinct crRNAs, these highly comprehensive CRISPRi libraries produced varying degrees of transcriptional repression critical for uncovering novel antibiotic resistance determinants. Furthermore, by iterating CRISPR adaptation, we rapidly generated dual-crRNA libraries representing more than 100,000 dual-gene perturbations. The polarized nature of spacer adaptation revealed the historical contingency in the stepwise acquisition of genetic perturbations leading to increasing antibiotic resistance. CALM circumvents the expense, labor, and time required for synthesis and cloning of gRNAs, allowing generation of CRISPRi libraries in wild-type bacteria refractory to routine genetic manipulation.

Protein kinase C (PKC) isozymes are members of the Serine/Threonine kinase family regulating cellular events following activation of membrane bound phospholipids. The breakdown of the downstream signaling pathways of PKC relates to several disease pathogeneses particularly neurodegeneration. PKC isozymes play a critical role in cell death and survival mechanisms, as well as autophagy. Numerous studies have reported that neurodegenerative disease formation is caused by failure of the autophagy mechanism. This review outlines PKC signaling in autophagy and neurodegenerative disease development and introduces some polyphenols as effectors of PKC isozymes for disease therapy.

<i>Background:</i> The interaction between obesity and genetic traits on high density lipoprotein (HDL) levels has been extensively studied. The variance of serum HDL has a strong genetic heritability, although the studied variant only explains a small part of this variation. The goal of this study was to investigate the associations between the apolipoprotein type 2 (<i>APOA2)</i> rs3813627 single nucleotide polymorphism (SNP) and anthropometric and biochemical variables, though body mass index (BMI). <i>Methods:</i> This study included 153 subjects (91 overweight/obese (BMI³25 kg/m²) and 62 non-obese individuals (BMI < 25 kg/m²)). The <i>APOA2</i> rs3813627 SNP was selected and genotyped. Genotype analysis was performed to analyze the associations between <i>APOA2</i> SNPs and anthropometric and biochemical variables through BMI. <i>Results:</i> The <i>APOA2</i> rs3813627 TT genotype was associated with low HDL levels in comparison with the <i>APOA2</i> rs3813627 GG and GT genotype in overweight/obese individuals, but not in the non-obese subjects (<i>p</i> < 0.05). The same trend was observed in the apolipoprotein type 1 (APOA1) protein levels (<i>p</i> < 0.05). Correlation analysis revealed a negative correlation between HDL and APOA1 levels and <i>APOA2</i> rs3813627 SNP under recessive model (<i>p</i> < 0.05). The odds ratio for low HDL levels was 3.76 and 3.94 for low APOA1 levels. The mediation analysis of <i>APOA2</i> rs3813627 SNP through BMI showed a full mediation on HDL and partial mediation on APOA1 levels (<i>p</i> < 0.05). Bioinformatic analysis showed that rs3813627 lies in the <i>APOA2</i> promoter and overlaps motifs for several bound transcription factors. <i>Conclusion</i>: On the basis of these data, the <i>APOA2</i> rs3813627 SNP is associated with low HDL and APOA1 levels susceptibility, and this effect was mediated by an increased BMI.

Suicide is a major public health concern; nevertheless, its neurobiology remains unknown. An area of interest in suicide research is the dorsolateral prefrontal cortex (DLPFC). We aimed to identify altered proteins and potential biological pathways in the DLPFC of individuals who died by suicide employing mass spectrometry-based untargeted proteomics. Postmortem DLPFC from age-matched male suicide mood disorder cases (n = 5) and non-suicide mood disorder cases (n = 5) were compared. The proteins that differed between groups at false discovery rate (FDR) adjusted p-values (Benjamini-Hochberg-Yekutieli) <0.3 and Log₂ fold change (FC) >|0.4| were considered statistically significant and were subjected to pathway analysis by Qiagen Ingenuity software. Thirty-three of the 5162 detected proteins showed significantly altered expression levels in the suicide cases and two of them after adjustment for body mass index. The top differentially expressed protein was potassium voltage-gated channel subfamily Q member 3 (KCNQ3) (Log₂FC = -0.481, <i>p</i> = 2.10 × 10^-09, FDR = 5.93 × 10^-06), which also showed a trend to downregulation in Western blot (<i>p</i> = 0.045, Bonferroni adjusted p = 0.090). The most notably enriched pathway was the GABA receptor signaling pathway (<i>p</i> < 0.001). Here, we report a reduction trend of KCNQ3 levels in the DLPFC of male suicide victims with mood disorders. Further studies with a larger sample size and equal sex representation are needed.

Type 2 diabetes mellitus (T2DM) is still a global health problem. Current T2DM treatments are limited to curing the symptoms and have not been able to restore insulin sensitivity in insulin-sensitive tissues that have become resistant. In the past decade, some studies have shown the significant role of a chaperone family, heat shock protein 70 (HSP70), in insulin resistance pathogenesis that leads to T2DM. HSP70 is a cytoprotective molecular chaperone that functions in protein folding and degradation. In general, studies have shown that decreased concentration of HSP70 is able to induce inflammation process through JNK activation, inhibit fatty acid oxidation by mitochondria through mitophagy decrease and mitochondrial biogenesis, as well as activate SREBP-1c, one of the lipogenic gene transcription factors in ER stress. The overall molecular pathways are potentially leading to insulin resistance and T2DM. Increased expression of HSP70 in brain tissues is able to improve insulin sensitivity and glycemic control specifically. HSP70 modulation-targeting strategies (including long-term physical exercise, hot tub therapy (HTT), and administration of alfalfa-derived HSP70 (aHSP70)) in subjects with insulin resistance are proven to have therapeutic and preventive potency that are promising in T2DM management.

In the present work, a theoretical study was carried out to study the molecular structure, harmonic vibrational frequencies, normal force field calculations, and Raman scattering activities for fluorene π-conjugation spacer containing azo-based dye named <i>trans</i>- and <i>cis</i>-bis(9<i>H</i>-fluoren-2-yl)diazene (AzoFL) at density functional theory using B3LYP (Becke-3-Lee-Yang-Parr) functional and 6-31+G(d,p) basis set. The theoretical calculations have also been performed with fluorene and the trans- and cis-isomers of diazene, difluorodiazene by the same method DFT-B3LYP/6-31+G(d,p) and basis set. The present DFT calculation shows that the <i>trans</i>-AzoFL is more stable than the <i>cis</i>-AzoFL by 16.33 kcal/mol. We also report the results of new assignments of vibrational frequencies obtained on the basis of the present calculations. Time-dependent DFT (TD-DFT) and ZIndo calculations have been performed to study the UV-vis absorption behavior and frontier molecular orbitals for the above-mentioned compounds. The UV-vis spectrum from TD-DFT calculation shows the π-π* transition bands at λ_max 423.53 nm (ε_max 6.0 × 10⁴ M^-1 cm^-1) and at λ_max 359.45 nm (ε_max 1.7 × 10⁴ M^-1 cm^-1), respectively, for <i>trans</i>- and <i>cis</i>-AzoFL. Compared to parent <i>trans</i>-diazene (λ_max 178.97 nm), a significant variation to longer wavelength (∼245 nm) is observed due to the incorporation of the fluorene (FL) ring into the -N=N- backbone. The co-planarity of the two FL rings with the longer N=N bond length compared to the unsubstituted parent diazene indicates the effective red shift due to the extended π-conjugation in <i>trans</i>-AzoFL. The nonplanarity of <i>cis</i>-AzoFL (48.1° tilted about the C-N bond relative to the planar N=N-C bond) reflects its ∼64 nm blue shift compared to that of trans-counterpart.

Hepatocellular carcinoma, a disease of the developing world, is known to present with extrahepatic metastases. Most common site being the lungs, it is not uncommon for metastases to present at unusual sites like the rectum, spleen and the diaphragm, among others. Metastases to the oral cavity is rare, with the most common primaries being lung, breast and the kidney. Metastases of a hepatocellular carcinoma to the oral cavity is a rare entity with extremely limited data in literature. We present one such unique case of oral cavity metastases from a hepatocellular carcinoma who presented to the Division of Head and Neck Oncology services of our hospital with a large oral cavity lesion, on subsequent workup of which, a hepatocellular carcinoma was identified. Awareness of this possibility can aid in accurate diagnosis and early management of a condition associated with an advanced stage at presentation and poorer prognosis.

<h4>Aims</h4>To analyze the main etiological factors and some clinical features of patients with hepatocellular carcinoma (HCC) at diagnosis and to compare them with those we described ten years ago. <i>Materials and Methods</i>. We compared two groups of patients with HCC, Group 1 consisting of 132 patients (82 M, 50 F) diagnosed in the 2003-2008 period and Group 2 including 119 patients (82 M, 37 F) diagnosed in the 2013-2018 period. For all patients, age, sex, viral markers, alcohol consumption, serum alpha-fetoprotein (AFP) levels, and the main liver function parameters were recorded. The diagnosis of HCC was based on AASLD, EASL guidelines. The staging was classified according to the "Barcelona Clinic Liver Cancer staging system" (BCLC).<h4>Results</h4>Mean age was 69.0 ± 8 years in Group 1 and 71.0 ± 9 in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (<i>P</i> < 0.05). HCV subjects were significantly older in Group 2 (.<h4>Conclusions</h4>This study shows that over the last decade a number of features of patients with HCC in our region have changed, particularly age at onset, etiological factors, and staging of HCC.

<h4>Background</h4>High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian carcinoma, associated with poor clinical outcome and metastatic disease. Although metastatic processes are becoming more understandable, the genomic landscape and metastatic progression in HGSOC has not been elucidated.<h4>Methods</h4>Multi-region whole-exome sequencing was performed on HGSOC primary tumours and their metastases (n = 33 tumour regions) from six patients. The resulting somatic variants were analysed to delineate tumour evolution and metastatic dissemination, and to compare the repertoire of events between primary HGSOC and metastasis.<h4>Results</h4>All cases presented branching evolution patterns in primary HGSOC, with three cases further showing parallel evolution in which different mutations on separate branches of a phylogenetic tree converge on the same gene. Furthermore, linear metastatic progression was observed in 67% of cases with late dissemination, in which the metastatic tumour mostly acquires the same mutational process active in primary tumour, and parallel metastatic progression, with early dissemination in the remaining 33.3% of cases. Metastatic-specific SNVs were further confirmed as late dissemination events. We also found the involvement of metastatic-specific driver events in the Wnt/β-catenin pathway, and identified potential clinically actionable events in individual patients of the metastatic HGSOC cohort.<h4>Conclusions</h4>This study provides deeper insights into clonal evolution and mutational processes that can pave the way to new therapeutic targets.

The faithful propagation of cellular signals in most organisms relies on the coordinated functions of a large family of protein kinases that share a conserved catalytic domain. The catalytic domain is a dynamic scaffold that undergoes large conformational changes upon activation. Most of these conformational changes, such as movement of the regulatory αC-helix from an "out" to "in" conformation, hinge on a conserved, but understudied, loop termed the αC-β4 loop, which mediates conserved interactions to tether flexible structural elements to the kinase core. We previously showed that the αC-β4 loop is a unique feature of eukaryotic protein kinases. Here, we review the emerging roles of this loop in kinase structure, function, regulation, and diseases. Through a kinome-wide analysis, we define the boundaries of the loop for the first time and show that sequence and structural variation in the loop correlate with conformational and regulatory variation. Many recurrent disease mutations map to the αC-β4 loop and contribute to drug resistance and abnormal kinase activation by relieving key auto-inhibitory interactions associated with αC-helix and inter-lobe movement. The αC-β4 loop is a hotspot for post-translational modifications, protein-protein interaction, and Hsp90 mediated folding. Our kinome-wide analysis provides insights for hypothesis-driven characterization of understudied kinases and the development of allosteric protein kinase inhibitors.

In mitosis, while the importance of kinetochore (KT)-microtubule (MT) attachment has been known for many years, increasing evidence suggests that telomere dysfunctions also perturb chromosome segregation by contributing to the formation of chromatin bridges at anaphase. Recent evidence suggests that Aurora B kinase ensures proper chromosome segregation during mitosis not only by controlling KT-MT attachment but also by regulating telomere and chromosome arm separation. However, whether and how Aurora B governs telomere separation during meiosis has remained unknown. Here, we show that fission yeast Aurora B localizes at telomeres during meiosis I and promotes telomere separation independently of the meiotic cohesin Rec8. In meiosis II, Aurora B controls KT-MT attachment but appears dispensable for telomere and chromosome arm separation. Likewise, condensin activity is nonessential in meiosis II for telomere and chromosome arm separation. Thus, in meiosis, the requirements for Aurora B are distinct at centromeres and telomeres, illustrating the critical differences in the control of chromosome segregation between mitosis and meiosis II.

Polybrominated diphenyl ethers (PBDEs) were formally used as flame-retardants and are chemically stable, lipophlic persistent organic pollutants which are known to bioaccumulate in humans. Although its toxicities are well characterized, little is known about the changes in transcriptional regulation caused by PBDE exposure. Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of transcriptional and translational processes. It is hypothesized that lncRNAs can regulate nearby protein-coding genes (PCGs) and changes in the transcription of lncRNAs may act in cis to perturb gene expression of its neighboring PCGs. The goals of this study were to 1) characterize PCGs and lncRNAs that are differentially regulated from exposure to PBDEs; 2) identify PCG-lncRNA pairs through genome annotation and predictive binding tools; and 3) determine enriched canonical pathways caused by differentially expressed lncRNA-PCGs pairs. HepaRG cells, which are human-derived hepatic cells that accurately represent gene expression profiles of human liver tissue, were exposed to BDE-47 and BDE-99 at a dose of 25 μM for 24 hours. Differentially expressed lncRNA-PCG pairs were identified through DESeq2 and HOMER; significant canonical pathways were determined through Ingenuity Pathway Analysis (IPA). LncTar was used to predict the binding of 19 lncRNA-PCG pairs with known roles in drug-processing pathways. Genome annotation revealed that the majority of the differentially expressed lncRNAs map to PCG introns. PBDEs regulated overlapping pathways with PXR and CAR such as protein ubiqutination pathway and peroxisome proliferator-activated receptor alpha-retinoid X receptor alpha (PPARα-RXRα) activation but also regulate distinctive pathways involved in intermediary metabolism. PBDEs uniquely down-regulated GDP-L-fucose biosynthesis, suggesting its role in modifying important pathways involved in intermediary metabolism such as carbohydrate and lipid metabolism. In conclusion, we provide strong evidence that PBDEs regulate both PCGs and lncRNAs in a PXR/CAR ligand-dependent and independent manner.

Vertebrate fossils embedded in amber represent a particularly valuable paleobiological record as amber is supposed to be a barrier to the environment, precluding significant alteration of the animals' body over geological time. The mode and processes of amber preservation are still under debate, and it is questionable to what extent original material may be preserved. Due to their high value, vertebrates in amber have never been examined with analytical methods, which means that the composition of bone tissue in amber is unknown. Here, we report our results of a study on a left forelimb from a fossil Anolis sp. indet. (Squamata) that was fully embedded in Miocene Dominican amber. Our results show a transformation of the bioapatite to fluorapatite associated with a severe alteration of the collagen phase and the formation of an unidentified carbonate. These findings argue for a poor survival potential of macromolecules in Dominican amber fossils.

We determined the clinical signs and blood ionized calcium (iCa) levels in dairy cows with peracute coliform mastitis (PCM). The clinical scores at the onset of the disease (day 0) and on day 2 and subsequent days were significantly (P<0.01) higher than those of healthy cows. We found a positive correlation (r=0.894, P<0.01) between iCa and total calcium (TCa) concentrations in the blood of healthy cows ; however there was no correlation from day 0 to day 3 in the blood of PCM cows. Multiple regression analysis revealed that the concentration of iCa was correlated with rectal temperature, hematocrit value, platelet count, and albumin level of PCM cows at the onset of disease (r= -0.804, r=0.6576, r=0.6182, r=0.284, P<0.01, respectively). There was no correlation between the TCa concentration and these parameters for PCM cows at day 0. Low blood iCa concentration at day 0 for PCM cows was related to symptoms of septic shock involving hypothermia, activation of the blood coagulation system, and dehydration.

<h4>Background</h4>Pulmonary malignant neoplasms have a high worldwide morbidity and mortality, so the study of these malignancies using microRNAs (miRNAs) has attracted great interest and enthusiasm. The aim of this study was to determine the clinical effect of hsa-microRNA-204-5p (miR-204-5p) and its underlying molecular mechanisms in non-small cell lung cancer (NSCLC).<h4>Methods</h4>Expression of miR-204-5p was investigated by real-time quantitative PCR (RT-qPCR). After data mining from public online repositories, several integrative assessment methods, including receiver operating characteristic (ROC) curves, hazard ratios (HR) with 95% confidence intervals (95% CI), and comprehensive meta-analyses, were conducted to explore the expression and clinical utility of miR-204-5p. The potential objects regulated and controlled by miR-204-5p in the course of NSCLC were identified by estimated target prediction and analysis. The regulatory network of miR-204-5p, with its target genes and transcription factors (TFs), was structured from database evidence and literature references.<h4>Results</h4>The expression of miR-204-5p was downregulated in NSCLC, and the downtrend was related to gender, histological type, vascular invasion, tumor size, clinicopathologic grade and lymph node metastasis (P<0.05). MiR-204-5p was useful in prognosis, but was deemed unsuitable at present as an auxiliary diagnostic or prognostic risk factor for NSCLC due to the lack of statistical significance in meta-analyses and absence of large-scale investigations. Gene enrichment and annotation analyses identified miR-204-5p candidate targets that took part in various genetic activities and biological functions. The predicted TFs, like MAX, MYC, and RUNX1, interfered in regulatory networks involving miR-204-5p and its predicted hub genes, though a modulatory loop or axis of the miRNA-TF-gene that was out of range with shortage in database prediction, experimental proof and literature confirmation.<h4>Conclusions</h4>The frequently observed decrease in miR-204-5p was helpful for NSCLC diagnosis. The estimated target genes and TFs contributed to the anti-oncogene effects of miR-204-5p.

Respiratory Distress Syndrome (RDS) is the commonest diagnosis after premature birth. We aimed to audit clinical practices before and after introduction of a national guideline in Wales on RDS management. Anonymised, prospective data on all infants born at <34 weeks of gestation and cared for at one of the participating neonatal units in Wales were collected in two six-month time periods in 2015 and 2018. A national guideline was introduced in 2016 by the Wales Neonatal Network. Data collection included areas of antenatal management, delivery room stabilisation, invasive and non-invasive respiratory support, surfactant treatment and elements of supportive care. Univariate and multivariate methods were used to compare data between the two epochs. Comparing care before and after introduction of the national guideline, areas of significant improvement include use of targeted tidal volume ventilation, use of caffeine therapy, oxygen therapy post-surfactant and increasing early use of parenteral nutrition. Areas of poorer management included levels of positive end expiratory pressures and timing of introduction of enteral feeds. Little variation was seen between level two and three units, although gestational age was a significant independent variable for several practices, including delayed cord clamping, stabilisation with intubation, early enteral feeding and caffeine administration. A national guideline for management of RDS in Wales has significantly improved practice in several areas. However, despite a large volume of high-quality evidence and robust guidance, there remains a significant variation in some elements of best practice for RDS management. Further work should focus on education and training, especially for elements requiring cross-departmental work.

Atopic dermatitis (AD) is a chronic, inflammatory skin disease that persists or repeatedly recurs in both childhood and adulthood. <i>Urtica thunbergiana</i> (UT) is an aroma herb with little-known pharmacological effects and anti-inflammatory activities against AD. This study investigated the immunomodulatory efficacy of 50% ethanol-extracted UT in necrosis factor-alpha/interferon-gamma (TNF-α/IFN-γ)-stimulated HaCaT cells in vitro and AD-Biostir-induced NC/Nga mice in vivo. The results showed that UT exhibits a dose-dependent increase in scavenged free radicals, reaching 76.0% ± 1.4% of scavenged 1,1-diphenyl-2-picrylhydrazyl at a concentration of 250 µg/mL. In addition, UT significantly downregulated the mRNA expression of the following pro-inflammatory cytokines and chemokines in TNF-α/IFN-γ-stimulated HaCaT cells: interleukin (IL)-6, IL-8, thymus- and activation-regulated chemokine, macrophage-derived chemokine, and regulated on activation normal T expressed and secreted. UT-treated HaCaT cells showed inhibition of the overexpression of chemokine-regulated signaling molecules, such as nuclear factor-kappa B, inhibitor of kappa B (IκBα), signal transducer and activator of transcription 1, and mitogen-activated protein kinases (MAPKs). UT dietary administration in AD-Biostir-induced NC/Nga mice treated and improved AD-like symptoms, such as scales, epidermal thickening, the dermatitis severity score, high trans-epidermal water loss, reduced skin hydration, increased mast cells, elevated serum immunoglobulin E levels, and an enlarged spleen. UT treatment inhibited the expression of phosphorylated forms of MAPKs, nuclear factor of activated T-cells 1, and regulator IκBα. It also upregulated filaggrin (FLG) production. Therefore, UT shows high anti-AD activity both in vitro and in vivo<i>,</i> and can be a useful anti-AD agent.

Angiogenesis is stimulated by nitric oxide (NO) production in endothelial cells (ECs). Although proangiogenic actions of human mesenchymal stem cells (hMSCs) have been extensively studied, the mechanistic role of NO in this action remains obscure. Here, we used a gelatin hydrogel that releases NO upon crosslinking by a transglutaminase reaction ("NO gel"). Then, the source-specific behaviors of bone marrow versus adipose tissue-derived hMSCs (BMSCs versus ADSCs) were monitored in the NO gels. NO inhibition resulted in significant decreases in their angiogenic activities. The NO gel induced pericyte-like characteristics in BMSCs in contrast to EC differentiation in ADSCs, as evidenced by tube stabilization versus tube formation, 3D colocalization versus 2D coformation with EC tube networks, pericyte-like wound healing versus EC-like vasculogenesis in gel plugs, and pericyte versus EC marker production. These results provide previously unidentified insights into the effects of NO in regulating hMSC source-specific angiogenic mechanisms and their therapeutic applications.

<h4>Purpose</h4>Cervical cancer is one of the most common causes of death among women globally. Combinations of cisplatin, paclitaxel, bevacizumab, carboplatin, topotecan, and gemcitabine are recommended as first-line therapies.<h4>Methods</h4>This study focuses on the development of folate-decorated, pH-sensitive lipid-polymer hybrid nanoparticles (LPNs). Loading carboplatin (CBP) and paclitaxel (PTX), LPNs were expected to combine the therapeutic effects of CBP and PTX, thus show synergistic ability on cervical cancer.<h4>Results</h4>FA-CBP/PTX-LPNs showed the sizes of 169.9 ± 5.6 nm, with a narrow size distribution of 0.151 ± 0.023. FA-CBP/PTX-LPNs exhibited pH-responsive drug release, high cellular uptake efficiency (66.7 ± 3.1%), and prominent cell inhibition capacity (23 ± 1.1%). In vivo tumor distribution and tumor inhibition efficiency of FA-CBP/PTX-LPNs was the highest, with no obvious body weight lost.<h4>Conclusion</h4>High tumor distribution and remarkable antitumor efficiency obtained using in vitro as well as in vivo models further proved the FA-CBP/PTX-LPNs is a promising tool for cervical cancer therapy.

<h4>Background</h4>Circular RNAs (circRNAs) play vital roles in the modulation of tumor progression. This study explored the biological functions of circ_0001105 in the progression of osteosarcoma (OS).<h4>Methods</h4>qRT-PCR and in situ hybridization (ISH) were performed to detect the expression status of circ_0001105 in cells and tissues. Bioinformatics analysis, dual-luciferase reporter gene assay, Western blot and qRT-PCR were performed to determine the relationships among RNAs. The CCK-8, colony formation, EdU, transwell and wound healing assays were conducted to evaluate the cell growth, invasion and migration of OS cells. Tumor xenografts were established to investigate the effects of circ_0001105 on tumor growth in vivo. Lastly, the protein expression of YTHDF2 in OS tissues was measured using immunohistochemical staining.<h4>Results</h4>Data showed that circ_0001105 and YTHDF2 were significantly lower, while miR-766 was higher in OS tissues compared to adjacent tissues. Low expression of circ_0001105 or YTHDF2 was associated with poor survival of OS patients as demonstrated by the Kaplan-Meier analysis. In addition, miR-766 was identified as a direct binding target of circ_0001105 and YTHDF2. Ectopic overexpression of circ_0001105 or YTHDF2 significantly suppressed OS cell viability and invasion through regulating miR-766. Last, overexpression of circ_0001105 significantly attenuated in vivo tumor growth.<h4>Conclusion</h4>Our findings suggest that circ_0001105 inhibits OS progression, at least partially, by regulating miR-766/YTHDF2 signaling pathway.

Blast exposure is common in military personnel during training and combat operations, yet biological mechanisms related to cell survival and function that coordinate recovery remain poorly understood. This study explored how moderate blast exposure influences gene expression; specifically, gene-network changes following moderate blast exposure. On day 1 (baseline) of a 10-day military training program, blood samples were drawn, and health and demographic information collected. Helmets equipped with bilateral sensors worn throughout training measured overpressure in pounds per square inch (psi). On day 7, some participants experienced moderate blast exposure (peak pressure ≥5 psi). On day 10, 3 days post-exposure, blood was collected and compared to baseline with RNA-sequencing to establish gene expression changes. Based on dysregulation data from RNA-sequencing, followed by top gene networks identified with Ingenuity Pathway Analysis, a subset of genes was validated (NanoString). Five gene networks were dysregulated; specifically, two highly significant networks: (1) Cell Death and Survival (score: 42), including 70 genes, with 50 downregulated and (2) Cell Structure, Function, and Metabolism (score: 41), including 69 genes, with 41 downregulated. Genes related to ubiquitination, including neuronal development and repair: UPF1, RNA Helicase and ATPase (<i>UPF1</i>) was upregulated while UPF3 Regulator of Nonsense Transcripts Homolog B (<i>UPF3B</i>) was downregulated. Genes related to inflammation were upregulated, including AKT serine/threonine kinase 1 (<i>AKT1</i>), a gene coordinating cellular recovery following TBIs. Moderate blast exposure induced significant gene expression changes including gene networks involved in (1) cell death and survival and (2) cellular development and function. The present findings may have implications for understanding blast exposure pathology and subsequent recovery efforts.

Four additives (Na₂WO₄, nano-hydroxyapatite, K₂TiF₆ and NaF) were added into the Na₅P₃O₁₀ + NaOH + C₃H₈O₃ base electrolyte according to the orthogonal design of four factors three levels (L₉ (3⁴)). Nine different micro-arc oxidation (MAO) coatings were fabricated on Mg-2Zn-0.5Ca alloys through orthogonal experiments. The effects of four additives on the microstructure, mechanical properties, corrosion resistance and biocompatibility of MAO coatings were investigated through X-ray diffraction (XRD), scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS), electrochemical corrosion test and <i>in vitro</i> degradation test. The addition of nano-hydroxyapatite and K₂TiF₆ showed self-sealing effects and contributed to the corrosion resistance of the samples significantly. The addition of 0.5 g L^-1 Na₂WO₄ markedly elevated the bonding strength of the coatings with the substrate. The optimal combination of factors and levels considering both mechanical properties and corrosion resistance was: 0.5 g L^-1 Na₂WO₄, 0 g L^-1 NaF, 5 g L^-1 n-HAp, 5 g L^-1 K₂TiF₆. The growth mechanism of MAO coatings combining with the visual phenomenon was discussed as well.

<h4>Scope</h4>To predict gut microbial metabolism of xenobiotics and the resulting plasma concentrations of metabolites formed, an in vitro-in silico-based testing strategy is developed using the isoflavone daidzein and its gut microbial metabolite S-equol as model compounds.<h4>Methods and results</h4>Anaerobic rat fecal incubations are optimized and performed to derive the apparent maximum velocities (V_max ) and Michaelis-Menten constants (K_m ) for gut microbial conversion of daidzein to dihydrodaidzein, S-equol, and O-desmethylangolensin, which are input as parameters for a physiologically based kinetic (PBK) model. The inclusion of gut microbiota in the PBK model allows prediction of S-equol concentrations and slightly reduced predicted maximal daidzein concentrations from 2.19 to 2.16 µm. The resulting predicted concentrations of daidzein and S-equol are comparable to in vivo concentrations reported.<h4>Conclusion</h4>The optimized in vitro approach to quantify kinetics for gut microbial conversions, and the newly developed PBK model for rats that includes gut microbial metabolism, provide a unique tool to predict the in vivo consequences of daidzein microbial metabolism for systemic exposure of the host to daidzein and its metabolite S-equol. The predictions reveal a dominant role for daidzein in ERα-mediated estrogenicity despite the higher estrogenic potency of its microbial metabolite S-equol.

DCC netrin 1 receptor (DCC) affects the structure and function of the dopamine circuitry, which in turn affects the susceptibility to developing addiction. In a previous study, we found that single nucleotide polymorphism (SNP) rs12607853 in the 3' untranslated region (3'-UTR) of DCC was significantly associated with heroin addiction. In the current study, we first used bioinformatics prediction to identify the DCC rs12607853 C allele as a potential hsa-miR-422a and hsa-miR-378c target site. We then used vector construction and dual-luciferase reporter assays to investigate the targeting relationship of DCC rs12607853 with hsa-miR-422a and hsa-miR-378c. The dual-luciferase reporter gene assay confirmed that the C allele of rs12607853 in combination with hsa-miR-422a led to repressed dual-luciferase gene expression. Moreover, gene expression assays disclosed that hsa-miR-422a inhibited DCC expression at both the mRNA and protein levels. We also found that morphine inhibited the expression of hsa-miR-422a but increased the expression of DCC mRNA, and this change in the expression of hsa-miR-422a could not be reversed by naloxone, which suggested that the role of DCC in opioid addiction might be regulated by hsa-miR-422a. In summary, this study improves our understanding of the role of hsa-miR-422a and identifies the genetic basis of rs12607853, which might contribute to the discovery of new biomarkers or therapeutic targets for opioid addiction.

<h4>Background</h4>Radioresistance of some non-small cell lung cancer (NSCLC) types increases the risk of recurrence or metastasis in afflicted patients, following radiotherapy. As such, further improvements to NSCLC radiotherapy are needed. The expression of oncogene TP53-regulated inhibitor of apoptosis 1 (TRIAP1) in NSCLC is increased following irradiation. Furthermore, gene set enrichment analysis (GSEA) has suggested that TRIAP1 might be involved in maintaining redox homeostasis. This in turn might enhance cell radioresistance.<h4>Methods</h4>In this study we irradiated human NSCLC cell lines (A549 and H460), while knocking down TRIAP1, to determine whether a disrupted redox homeostasis could attenuate radioresistance.<h4>Results</h4>Irradiation notably increased both mRNA and protein levels of TRIAP1. In addition, TRIAP1 knockdown decreased the expression of several antioxidant proteins, including thioredoxin-related transmembrane protein (TMX) 1, TMX2, thioredoxin (TXN), glutaredoxin (GLRX) 2, GLRX3, peroxiredoxin (PRDX) 3, PRDX4, and PRDX6 in A549 and H460 cells. In addition, silencing TRIAP1 impaired the radiation-induced increase of the aforementioned proteins. Continuing along this line, we observed a radiation-induced reduction of cell viability and invasion, as well as increased apoptosis and intracellular reactive oxygen species following TRIAP1 knockdown.<h4>Conclusions</h4>In summary, we identified TRIAP1 as a key contributor to the radioresistance of NSCLC by maintaining redox homeostasis.

Glucocorticoids (GC) are the foundation of the chemotherapy regimen in acute lymphoblastic leukemia (ALL). However, resistance to GC is observed more frequently than resistance to other chemotherapy agents in patients with ALL relapse. Moreover, the mechanism underlying the development of GC resistance in ALL has not yet been fully uncovered. In this study, we used bioinformatic analysis methods to integrate the candidate genes and pathways participating in GC resistance in ALL and subsequently verified the bioinformatics findings with in vitro cell experiments. Ninety-nine significant common differentially expressed genes (DEGs) associated with GC resistance were determined by integrating two gene profile datasets, including GC-sensitive and -resistant samples. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathways analysis, the signaling pathways in which DEGs were significantly enriched were clustered. The GC resistance-related biologically functional interactions were visualized as DEG-associated Protein-Protein Interaction (PPI) network complexes, with 98 nodes and 127 edges. MYC, a node which displayed the highest connectivity in all edges, was highlighted as the core gene in the PPI network. Increased C-MYC expression was observed in adriamycin-resistant BALL-1/ADR cells, which we demonstrated was also resistant to dexamethasone. These results outlined a panorama in which the solitary and scattered experimental results were integrated and expanded. The potential promising target of the candidate pathways and genes involved in GC resistance of ALL was concomitantly revealed.

The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as <i>Dcc</i> knockouts die at birth. To circumvent this drawback, we generated an eye-specific <i>Dcc</i> mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of <i>Dcc</i>, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, <i>Dcc</i>-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.

T cell anergy is known to be a crucial mechanism for various types of immune tolerance, including oral tolerance. The expression of several anergy-specific genes was reportedly up-regulated in anergic T cells, and played important roles in the cells. However, how the genes were up-regulated has not been understood. In this study, we comprehensively analyzed the altered gene expression and DNA methylation status in T cells tolerized by oral antigen in vivo. Our results showed that many genes were significantly up-regulated in the orally tolerized T cells, and most of the genes found in this study have not been reported previously as anergy related genes; for example, ribosomal protein L41 (FC = 3.54E06, p = 3.70E-09: Fisher's exact test; the same applies hereinafter) and CD52 (FC = 2.18E05, p = 3.44E-06). Furthermore, we showed that the DNA methylation statuses of many genes; for example, enoyl-coenzyme A delta isomerase 3 (FC = 3.62E-01, p = 3.01E-02) and leucine zipper protein 1 (FC = 4.80E-01, p = 3.25E-02), including the ones distinctly expressed in tolerized T cells; for example, latexin (FC = 3.85E03, p = 4.06E-02 for expression; FC = 7.75E-01, p = 4.13E-01 for DNA methylation) and small nuclear ribonucleoprotein polypeptide F (FC = 3.12E04, p = 4.46E-04 for expression; FC = 8.56E-01, p = 5.15E-01 for DNA methylation), changed during tolerization, suggesting that the distinct expression of some genes was epigenetically regulated in the tolerized T cells. This study would contribute to providing a novel clue to the fine understanding of the mechanism for T cell anergy and oral tolerance.

<h4>Introduction</h4>Interaction of OX40 and OX40 ligand (OX40L) is associated with immune activation. OX40-OX40L axis is also suggested to play a role in immunity against several solid malignancies.<h4>Objective</h4>In this study, serum OX40 and OX40L levels in patients with advanced lung adenocarcinoma were assessed and their correlation with survival and clinicopathologic parameters was determined.<h4>Methods</h4>Serum samples were collected from patients with advanced lung adenocarcinoma, then OX40 and OX40L were quantified via enzyme-linked immunosorbent assay. Immunohistochemical (IHC) analysis of OX40 and OX40L in resected primary lesions was also performed. The association between OX40 and OX40L levels and clinicopathologic status and patient survival was retrospectively analyzed.<h4>Results</h4>A total of 56 patients were analyzed. Median serum OX40 and OX40L levels were 156.2 pg/mL and 186.6 pg/mL, respectively. IHC analysis in 5 patients indicated high positivity of OX40 in tumor-infiltrating lymphocytes and of OX40L in tumor cells in mucinous adenocarcinoma. Patients with a high OX40 level (≥152.2 pg/mL) had poorer prognosis than those with a low serum OX40 level (median survival, 7.36 vs. 21.19 months, respectively, p = 0.04). Patients with a high OX40L level (≥207.3 pg/mL) had poorer prognosis than those with a low serum OX40L level (median survival, 7.36 vs. 14.26 months, respectively, p = 0.04). In the subset of patients treated with immune checkpoint inhibitors (ICIs) (n = 12), those with a high OX40L level were found to have longer survival from ICI initiation than those with a low OX40L level (p = 0.023).<h4>Conclusions</h4>High OX40 and OX40L levels are associated with poor prognosis and may reflect the immune-exhausted status against lung adenocarcinoma.

<h4>Purpose</h4>To determine if the degree of estrogen suppression with aromatase inhibitors (AI: anastrozole, exemestane, letrozole) is associated with efficacy in early-stage breast cancer, and to examine for differences in the mechanism of action between the three AIs.<h4>Experimental design</h4>Matched case-control studies [247 matched sets from MA.27 (anastrozole vs. exemestane) and PreFace (letrozole) trials] were undertaken to assess whether estrone (E1) or estradiol (E2) concentrations after 6 months of adjuvant therapy were associated with risk of an early breast cancer event (EBCE). Preclinical laboratory studies included luciferase activity, cell proliferation, radio-labeled ligand estrogen receptor binding, surface plasmon resonance ligand receptor binding, and nuclear magnetic resonance assays.<h4>Results</h4>Women with E1 ≥1.3 pg/mL and E2 ≥0.5 pg/mL after 6 months of AI treatment had a 2.2-fold increase in risk (<i>P</i> = 0.0005) of an EBCE, and in the anastrozole subgroup, the increase in risk of an EBCE was 3.0-fold (<i>P</i> = 0.001). Preclinical laboratory studies examined mechanisms of action in addition to aromatase inhibition and showed that only anastrozole could directly bind to estrogen receptor α (ERα), activate estrogen response element-dependent transcription, and stimulate growth of an aromatase-deficient <i>CYP19A1^-/-</i> T47D breast cancer cell line.<h4>Conclusions</h4>This matched case-control clinical study revealed that levels of estrone and estradiol above identified thresholds after 6 months of adjuvant anastrozole treatment were associated with increased risk of an EBCE. Preclinical laboratory studies revealed that anastrozole, but not exemestane or letrozole, is a ligand for ERα. These findings represent potential steps towards individualized anastrozole therapy.

It is commonly assumed that methane (CH₄) released by lakes into the atmosphere is mainly produced in anoxic sediment and transported by diffusion or ebullition through the water column to the surface of the lake. In contrast to that prevailing idea, it has been gradually established that the epilimnetic CH₄ does not originate exclusively from sediments but is also locally produced or laterally transported from the littoral zone. Therefore, CH₄ cycling in the epilimnion and the hypolimnion might not be as closely linked as previously thought. We utilized a high-resolution method used to determine dissolved CH₄ concentration to analyze a Siberian lake in which epilimnetic and hypolimnetic CH₄ cycles were fully segregated by a section of the water column where CH₄ was not detected. This layer, with no detected CH₄, was well below the oxycline and the photic zone and thus assumed to be anaerobic. However, on the basis of a diffusion-reaction model, molecular biology, and stable isotope analyses, we determined that this layer takes up all the CH₄ produced in the sediments and the deepest section of the hypolimnion. We concluded that there was no CH₄ exchange between the hypolimnion (dominated by methanotrophy and methanogenesis) and the epilimnion (dominated by methane lateral transport and/or oxic production), resulting in a vertically segregated lake internal CH₄ cycle.

Despite advances in high-throughput sequencing that have revolutionized the discovery of gene defects in rare Mendelian diseases, there are still gaps in translating individual genome variation to observed phenotypic outcomes. While we continue to improve genomics approaches to identify primary disease-causing variants, it is evident that no genetic variant acts alone. In other words, some other variants in the genome (genetic modifiers) may alleviate (suppress) or exacerbate (enhance) the severity of the disease, resulting in the variability of phenotypic outcomes. Thus, to truly understand the disease, we need to consider how the disease-causing variants interact with the rest of the genome in an individual. Here, we review the current state-of-the-field in the identification of genetic modifiers in rare Mendelian diseases and discuss the potential for future approaches that could bridge the existing gap.

Amphotericin B (AmB) is a broad spectrum of antifungal drug used to treat antifungal diseases. However, due to the high toxicity of AmB, treated patients may suffer the risk of side effects, such as renal failure. Nanoencapsulation strategies have been reported to elicit low toxicity, albeit most of them possess low encapsulation efficiency. The aim of this research is to develop micellar delivery systems for AmB with reduced toxicity while maintaining its affectivity by employing retinol (RET)-conjugated amphiphilic block copolymers (ABCs) as precursors. Copolymers composed of poly(ε-caprolactone) (A) and polyethylenglycol (B) of types AB and ABA were synthesized by ring opening polymerization and subsequently conjugated with RET by Steglich esterification. ¹H-NMR spectroscopy was used to corroborate the structure of copolymers and their conjugates and determine their molecular weights. Analysis by gel permeation chromatography also found that the materials have narrow distributions. The resulting copolymers were used as precursors for delivery systems of AmB, thus reducing its aggregation and consequently causing a low haemolytic effect. Upon conjugation with RET, the encapsulation capacity was enhanced from approximately 2 wt % for AB and ABA copolymers to 10 wt %. AmB encapsulated in polymer micelles presented improved antifungal efficiency against <i>Candida</i> <i>albicans</i> and <i>C</i><i>andida</i> <i>auris</i> strains compared with Fungizone®, as deduced from the low minimum inhibitory concentration.

A Tepary bean lectin fraction (TBLF) has been studied because it exhibits differential cytotoxic and anticancer effects on colon cancer. The present work focuses on the evaluation of the apoptotic mechanism of action on colon cancer cells. Initially, lethal concentrations (LC₅₀) were obtained for the three studied cell lines (HT-29, RKO and SW-480). HT-29 showed the highest LC₅₀, 10 and 100 times higher than that of RKO and SW-480 cells, respectively. Apoptosis was evaluated by flow cytometry, where HT-29 cells showed the highest levels of early and total apoptosis, caspases activity was confirmed and necrosis was discarded. The effect on cell cycle arrest was shown in the G0/G1 phase. Specific apoptosis-related gene expression was determined, where an increase in p53 and a decrease in Bcl-2 were observed. Expression of p53 gene showed the maximum level at 8 h with an important decrease at 12 and 24 h, also the phosphorylated p53(ser46) increased at 8 h. Our results show that TBLF induces apoptosis in colon cancer cells by p-p53(ser46) involvement. Further studies will focus on studying the specific signal transduction pathway.

<h4>Objective</h4>Overweight and obesity are major risk factors for Type 2 Diabetes Mellitus (T2DM), cardiovascular disease (CVD) and cancer. Genetic predisposition has been shown to play a key role in obesity, and genome-wide association studies (GWAS) have identified multiple loci linked with obesity in various ethnic groups. The aim of this study was to validate the reported genetic variants associated with obesity and overweight in a young UAE Arab population.<h4>Methods</h4>Twenty-two associated single nucleotide polymorphisms (SNPs) at 11 loci (FTO, MC4R, TMEM18, KCTD15, MTCH2, SH2B1, TFAP2B, GNPDA2, NEGR1, PCSK1 and BDNF) were studied in 392 controls and 318 overweight/obese young Emiratis (aged 18-35 years).<h4>Results</h4>After adjusting for age and smoking, rs3751812 of the FTO gene was associated with overweight/obesity in male participants (p-value < 0.016), while SNPs rs17782313, rs571312 of the MC4R gene and rs12463617 of the TMEM18 gene were significantly associated with overweight/obesity in female participants (p-value = 0.001, 0.028, 0.044, respectively). Follow-up association tests and logistic regression revealed the contribution of the FTO rs3751812 and MC4R rs571213 SNPs to the risk of overweight/obesity after adjusting for age, sex and smoking (p-value = 0.044, 0.049, respectively). In addition, the FTO rs3751812 was associated with the risk of overweight/obesity after adjusting for the effect of other markers (rs17782313, rs571312, rs2867125, rs6548238 and rs12463617) (p-value = 0.035). A significant gene-gene interaction was seen between FTO, MCR4 and TMEM18 (p-value = 0.013).<h4>Conclusions</h4>Our data demonstrates that rs3751812 of the FTO gene is the key SNP associated with risk of overweight/obesity among the young UAE Arab population, in alignment with previous findings. Our results also indicate that the identified genes stratify with sex and risk of overweight/obesity. In addition to their direct association with overweight/obesity, rs17782313 and rs571312, as well as rs2867125 and rs6548238, may have a modifying effect on the risk of overweight/obesity caused by the rs3751812. Population-specific, sex-specific genetic profiling is important in understanding the heritability of obesity.

Forkhead Box G1 (<i>FOXG1</i>) is a member of the Forkhead family of genes with non-redundant roles in brain development, where alteration of this gene's expression significantly affects the formation and function of the mammalian cerebral cortex. <i>FOXG1</i> haploinsufficiency in humans is associated with prominent differences in brain size and impaired intellectual development noticeable in early childhood, while homozygous mutations are typically fatal. As such, <i>FOXG1</i> has been implicated in a wide spectrum of congenital brain disorders, including the congenital variant of Rett syndrome, infantile spasms, microcephaly, autism spectrum disorder (ASD) and schizophrenia. Recent technological advances have yielded greater insight into phenotypic variations observed in FOXG1 syndrome, molecular mechanisms underlying pathogenesis of the disease, and multifaceted roles of <i>FOXG1</i> expression. In this review, we explore the emerging mechanisms of <i>FOXG1</i> in a range of transcriptional to posttranscriptional events in order to evolve our current view of how a single transcription factor governs the assembly of an elaborate cortical circuit responsible for higher cognitive functions and neurological disorders.

The pathogenesis of Huntington's disease (HD), an inherited progressive neurodegenerative disease, is highly associated with the cytotoxicity-inducing mutant huntingtin (mHtt) protein. Emerging evidence indicates that autophagy plays a pivotal role in degrading aggregated proteins such as mHtt to enhance neuronal viability. In this study, by employing preparative high-performance liquid chromatography (pre-HPLC), ultra-high performance liquid chromatography diode-array-detector quadrupole time-of-flight mass spectrometry (UHPLC-DAD-Q-TOF-MS) and nuclear magnetic resonance (NMR), three escins, escin IA (EA), escin IB (EB) and isoescin IA (IEA), were isolated and identified from the seed of <i>Aesculus chinensis</i> Bge. (ACB). After EGFP-HTT74-overexpressing HT22 cells were treated with EA, EB and IEA at safe concentrations, the clearance of mHtt and mHtt-induced apoptosis were investigated by Western blot, immunofluorescence microscopy and flow cytometry methods. In addition, the autophagy induced by these escins in HT22 cells was monitored by detecting GFP-LC3 puncta, P62 and LC3 protein expression. The results showed that EA, EB and IEA could significantly decrease mHtt levels and inhibit its induced apoptosis in HT22 cells. In addition, these three saponins induced autophagic flux by increasing the ratio of RFP-LC3 to GFP-LC3, and by decreasing P62 expression. Among the tested escins, EB displayed the best autophagy induction, which was regulated <i>via</i> both the mTOR and ERK signaling pathways. Furthermore, the degradation of mHtt and the commensurate decrease in its cytotoxic effects by EA, EB and IEA were demonstrated to be closely associated with autophagy induction, which depended on ATG7. In conclusion, we are the first to report that the escins, including EA, EB and IEA are novel autophagy inducers that degrade mHtt and inhibit mHtt-induced apoptosis <i>in vitro</i> and <i>in vivo</i>. As a result of these findings, the triterpenoid saponins in ACB might be considered to be promising candidates for the treatment of HD in the future.

Electrospinning is a technique that uses polymer solutions and strong electric fields to produce nano-sized fibers that have wide-ranging applications. We present here an overview of the use of electrospinning to incorporate biological products into nanofibers, including microorganisms, cells, proteins, and nucleic acids. Although the conditions used during electrospinning limit the already problematic viability/stability of such biological products, their effective incorporation into nanofibers has been shown to be feasible. Synthetic polymers have been more frequently applied to make nanofibers than natural polymers. Polymer blends are commonly used to achieve favorable physical properties of nanofibers. The majority of nanofibers that contain biological product have been designed for therapeutic applications. The incorporation of these biological products into nanofibers can promote their stability or viability, and also allow their delivery to a desired tissue or organ. Other applications include plant protection in agriculture, fermentation in the food industry, biocatalytic environmental remediation, and biosensing. Live cells that have been incorporated into nanofibers include bacteria and fungi. Nanofibers have served as scaffolds for stem cells seeded on a surface, to enable their delivery and application in tissue regeneration and wound healing. Viruses incorporated into nanofibers have been used in gene delivery, as well as in therapies against bacterial infections and cancers. Proteins (hormones, growth factors, and enzymes) and nucleic acids (DNA and RNA) have been incorporated into nanofibers, mainly to treat diseases and enhance their stability. To summarize, incorporation of biological products into nanofibers has numerous advantages, such as providing protection and facilitating controlled delivery from a solid form with a large surface area. Future studies should address the challenge of transferring nanofibers with biological products into practical and industrial use.

Modulation of immune activation using immunotherapy has attracted considerable attention for many years as a potential therapeutic intervention for several inflammation-associated neurodegenerative diseases. However, the efficacy of single-target immunotherapy intervention has shown limited or no efficacy in alleviating disease burden and restoring functional capacity. Marked immune system activation and neuroinflammation are important features and prodromal signs in polyQ repeat disorders and α-synucleinopathies. This review describes the current status and future directions of immunotherapies in proteinopathy-induced neurodegeneration with emphasis on preclinical and clinical efficacies of several anti-inflammatory compounds and antibody-based therapies for the treatment of Huntington's disease and α-synucleinopathies. The review concludes with how disease modification and functional restoration could be achieved by using targeted multimodality therapy to target multiple factors.

<h4>Background and aims</h4>To study the clinical profile of genetically proven Huntington's disease (HD) patients from eastern India.<h4>Methods</h4>This cross sectional study selected patients of HD after genetic confirmation of expanded CAG repeats in Huntingtin (<i>HTT</i>) gene. We performed detail clinical evaluation including cognitive and neuropsychological assessment, and imaging of brain.<h4>Results</h4>This study included 75 patients (male: 57.3%; female: 42.7%). Mean age at onset was 37.12 (range 16-62) years; juvenile variety (onset below 20 years) was detected in 5.3%. Paternal transmission was commoner. Manifestations at onset were motor in 81.3% patients, behavioral in 10.7% and cognitive impairment in 8%. After chorea, next common movement disorder was dystonia. Frontal lobe dysfunction was found in 77.3% patients. Behavioral disturbances were observed in 77.3% patients and commonly manifested as depression, irritable behavior and anxiety. Among the three onset groups (motor/behavioral/cognitive), there was no significant difference regarding age at onset, gender distribution, pattern of inheritance (paternal/maternal), and at the time of evaluation, all groups had essentially similar pattern of clinical features. Mean CAG repeat of the patients was 48.25 (range 40-79). Our study showed some differing clinical characteristics compared to previous studies from the Indian subcontinent.<h4>Conclusion</h4>Clinical features in our study showed differences from previous studies from the Indian subcontinent. We had more cognitive-onset patients. However, behavioral onset was lower in our study. Motor, behavioral and cognitive onset groups of HD were comparable regarding demographics, family history, CAG repeat lengths and major clinical features at the time of evaluation.

Social epigenomics is a new field of research that studies how the social environment shapes the epigenome and how in turn the epigenome modulates behavior. We focus on describing known gene-environment interactions (GEIs) and epigenetic mechanisms in different mammalian social systems. To illustrate how epigenetic mechanisms integrate GEIs, we highlight examples where epigenetic mechanisms are associated with social behaviors and with their maintenance through neuroendocrine, locomotor, and metabolic responses. We discuss future research trajectories and open questions for the emerging field of social epigenomics in nonmodel and naturally occurring social systems. Finally, we outline the technological advances that aid the study of epigenetic mechanisms in the establishment of GEIs and vice versa.

Sepsis remains one of the most lethal and costly conditions treated in U.S. hospitals, with approximately 50% of cases caused by Gram-negative bacterial infections. Septic shock is induced when lipopolysaccharide (LPS), the main component of Gram-negative outer bacterial membrane, signals through the Toll-like receptor 4 (TLR4) complex. Lethal endotoxemia, a model for septic shock, was induced in WT C57BL6 and TLR4^-/- mice by administration of <i>Escherichia coli</i> LPS. WT LPS treated mice showed high morbidity, while PBS treated LPS and treated TLR4^-/- mice did not. ANOVA analysis of label-free quantification of longitudinal serum proteome revealed 182 out of 324 proteins in LPS injected WT mice that were significantly changed across four time points (0, 6, 12, and 18 h). No significant changes were identified in the two control groups. From the 182 identified proteins, examples of known sepsis biomarkers were validated by ELISA, which showed similar trends as MS proteomics data. Longitudinal analysis within individual mice produced 3-fold more significantly changed proteins than pair-wise comparison. A subsequent global analysis of WT and TLR4^-/- mice identified pathways activated independent of TLR4. These pathways represent possible compensatory mechanisms that allow for control of Gram-negative bacterial infection regardless of host immune status.

Embryonic and neonatal skeletal muscles grow via the proliferation and fusion of myogenic cells, whereas adult skeletal muscle adapts largely by remodelling pre-existing myofibers and optimizing metabolic balance. It has been reported that miRNAs played key roles during skeletal muscle development through targeting different genes at post-transcriptional level. In this study, we show that a single miRNA (miR-208b) can modulate both the myogenesis and homoeostasis of skeletal muscle by distinct targets. As results, miR-208b accelerates the proliferation and inhibits the differentiation of myogenic cells by targeting the E-protein family member transcription factor 12 (TCF12). Also, miR-208b can stimulate fast-to-slow fibre conversion and oxidative metabolism programme through targeting folliculin interacting protein 1 (FNIP1) but not TCF12 gene. Further, miR-208b could active the AMPK/PGC-1a signalling and mitochondrial biogenesis through targeting FNIP1. Thus, miR-208b could mediate skeletal muscle development and homoeostasis through specifically targeting of TCF12 and FNIP1.

Our aims were to 1) evaluate the capacity of hollow hydroxyapatite (HA) microspheres (212-250 μm) to serve as a delivery system for controlled release of BMP-2 in vitro and 2) examine relaxin as an enhancer of BMP-2 for bone regeneration. Hollow HA microspheres were converted from borate glass microspheres and characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and the Brunauer-Emmett-Teller method. The microspheres loaded with BMP-2 and relaxin were implanted for 6 weeks in Sprague Dawley rats with calvarial defects. BMP-2 alone in the range up to 1 μg per defect exhibited dose-dependent bone regeneration while relaxin alone in the range up to 0.25 μg per defect did not promote bone regeneration. When compared with BMP-2 alone (1 μg per defect), a 50% reduction in the BMP-2 dose was achieved with the addition of 0.05, 0.1, or 0.25 μg of relaxin per defect. These results show that loading HA microspheres with a combination of relaxin and BMP-2 can significantly reduce the BMP-2 dose required to regenerate an equivalent amount of bone.

<h4>Background</h4>Invasive hemodynamic evaluation through right heart catheterization plays an essential role in the diagnosis, categorization, and risk stratification of patients with pulmonary hypertension.<h4>Methods</h4>Subjects enrolled in the PVDOMICS (Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics) program undergo an extensive invasive hemodynamic evaluation that includes repeated measurements at rest and during several provocative physiological challenges. It is a National Institutes of Health/National Heart, Lung, and Blood Institute initiative to reclassify pulmonary hypertension groups based on clustered phenotypic and phenomic characteristics. At a subset of centers, participants also undergo an invasive cardiopulmonary exercise test to assess changes in hemodynamics and gas exchange during exercise.<h4>Conclusions</h4>When coupled with other physiological testing and blood -omic analyses involved in the PVDOMICS study, the comprehensive right heart catheterization protocol described here holds promise to clarify the diagnosis and clustering of pulmonary hypertension patients into cohorts beyond the traditional 5 World Symposium on Pulmonary Hypertension groups. This article will describe the methods applied for invasive hemodynamic characterization in the PVDOMICS program. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02980887.

Research on non-coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature.

Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.

A breakthrough in oncology over the last 5 years, immunotherapy has proved its salutary effects in a wide range of solid tumors. The targeting of the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway can restore a competent antitumor T-cell response by addressing key tumor immune evasion mechanisms. This novel mechanism of action is associated with new patterns of responses that were not observed with conventional treatments such as chemotherapy or targeted therapies. Thus, hyperprogressive disease (HPD), an unexpected acceleration of cancer evolution after starting immunotherapy, has been reported by several groups with a PD-1/PD-L1 blockade. This tumor flare-up phenomenon is associated with a poorer outcome and is suspected to be an immune-related adverse event. Despite been highly debated, the issue of HPD is currently a real challenge for oncologists' practice in terms of patients' information, diagnosis, and management. Herein, we describe the case of a 57-year-old man diagnosed with metastatic urothelial carcinoma who developed a rapid tumor growth after an anti-PD-L1+ IO combination. This case illustrates how current practice should evolve to address the HPD reality in the anticheckpoint era. KEY POINTS: Hyperprogressive disease (HPD) is an unexpected acceleration of cancer growth after starting immunotherapy that is associated with a poor outcome. Definition of HPD is based on comparing kinetics of tumor growth before and after starting immunotherapy. No predictive biomarker has been homogenously identified in the reported studies. Suspected pathophysiology includes expansion of programmed cell death protein 1 (PD-1) + regulatory T cells, exhaustion of compensatory T cells, modulation of pro-tumorigenic immune cell subsets, activation of aberrant inflammation, or activation of oncogenic signaling. HPD is one of the most controversial immune-related adverse events, as the liability of immunotherapy in this tumor deleterious flare-up phenomenon has not been proved yet. The reported incidence of HPD in retrospective studies varies across different solid tumor types from 6% to 29%. This phenomenon has been mainly suspected in non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and in urothelial carcinomas, where several randomized phase III trials have shown early crossing over of survival curves. In the context of anti-PD-1/programmed death-ligand 1 therapy, in particular for NSCLC, HNSCC, or urothelial carcinoma, the authors recommend performing an early computed tomography (CT) assessment at week 3-4. In the case of an early progression, tumor molecular characterization by tumor biopsy or circulating tumor DNA could be urged. Immunotherapy discontinuation should be discussed. Performing a confirmatory CT scan 4 weeks later to exclude pseudoprogression should not be the rule. Early switch to cytotoxic therapy may counteract the deleterious flare-up. Patients should be informed of the risk of developing HPD. Health authorities and trial sponsors could monitor and report the rates of tumor flares in trials in order to help oncologists to properly inform their patients about the expected rates of HPD.

Scedosporium species are opportunistic pathogens causing various infections, including disseminated infections in severely immunocompromised patients. Preventive measures aiming to reduce the risk of exposure to these fungi require a better knowledge on their ecology and on the sources of contamination of the patients. In this context, 99 soil samples from the Rabat-Sale-Kenitra and Fez-Meknes regions in Morocco were analyzed. Samples were inoculated on the highly selective Scedo-Select III culture medium, and seven chemical parameters of the soils were measured. Scedosporium species were detected in 48 of the samples, with the highest density in soils from wastewater treatment plants and landfills, followed by those from roadsides and polluted riverbanks, thus confirming the impact of human activities on their ecology. Scedosporium apiospermum was the most common species, followed by S. boydii and S. aurantiacum. Analysis of the chemical parameters of the soils revealed the presence of Scedosporium species was mainly associated with a moderate electrical conductivity, a pH range of 7.0 to 7.6, a nutrient-rich content and a moderate phosphorus amount. Thereby, these results demonstrated the relatively high occurrence of Scedosporium in Morocco and highlighted the impact of phosphorus content on their ecology.

The precise degradation of dysfunctional mitochondria by mitophagy is essential for maintaining neuronal homeostasis. HTT (huntingtin) can interact with numerous other proteins and thereby perform multiple biological functions within the cell. In this study, we investigated the role of HTT during mitophagy and analyzed the impact of the expansion of its polyglutamine (polyQ) tract. HTT is involved in different mitophagy steps, promoting the physical proximity of different protein complexes during the initiation of mitophagy and recruiting mitophagy receptors essential for promoting the interaction between damaged mitochondria and the nascent autophagosome. The presence of the polyQ tract in mutant HTT affects the formation of these protein complexes and determines the negative consequences of mutant HTT on mitophagy, leading to the accumulation of damaged mitochondria and an increase in oxidative stress. These outcomes contribute to general mitochondrial dysfunction and neurodegeneration in Huntington disease.<b>Abbreviations</b>: AMPK: AMP-activated protein kinase; ATG13: autophagy related 13; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene-diamine-tetra-acetic acid; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; FUNDC1: FUN14 domain containing 1; HD: Huntington disease; HRP: horseradish peroxidase; HTT: huntingtin; LC3-II: lipidated form of MAP1LC3/LC3; mtDNA: mitochondrial deoxyribonucleic acid; MTDR: MitoTracker Deep Red; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1, autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; OCR: oxygen consumption rate; OPTN: optineurin; OXPHOS: oxidative phosphorylation; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PINK1: PTEN induced putative kinase 1; PLA: proximity ligation assay; PMSF: phenylmethylsulfonyl fluoride; polyQ: polyglutamine; PtdIns3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; Rot: rotenone; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TMRM: tetramethylrhodamine methyl ester; UB: ubiquitin; ULK1: unc-51 like kinase 1.

<h4>Background</h4>MicroRNA (miRNA) regulation is associated with several diseases, including neurodegenerative diseases. Several approaches can be used for modeling miRNA regulation. However, their precision may be limited for analyzing multidimensional data. Here, we addressed this question by integrating shape analysis and feature selection into miRAMINT, a methodology that we used for analyzing multidimensional RNA-seq and proteomic data from a knock-in mouse model (Hdh mice) of Huntington's disease (HD), a disease caused by CAG repeat expansion in huntingtin (htt). This dataset covers 6 CAG repeat alleles and 3 age points in the striatum and cortex of Hdh mice.<h4>Results</h4>Remarkably, compared to previous analyzes of this multidimensional dataset, the miRAMINT approach retained only 31 explanatory striatal miRNA-mRNA pairs that are precisely associated with the shape of CAG repeat dependence over time, among which 5 pairs with a strong change of target expression levels. Several of these pairs were previously associated with neuronal homeostasis or HD pathogenesis, or both. Such miRNA-mRNA pairs were not detected in cortex.<h4>Conclusions</h4>These data suggest that miRNA regulation has a limited global role in HD while providing accurately-selected miRNA-target pairs to study how the brain may compute molecular responses to HD over time. These data also provide a methodological framework for researchers to explore how shape analysis can enhance multidimensional data analytics in biology and disease.

<h4>Background</h4>Genome-wide association studies (GWAS) provide a powerful means to identify associations between genetic variants and phenotypes. However, GWAS techniques for detecting epistasis, the interactions between genetic variants associated with phenotypes, are still limited. We believe that developing an efficient and effective GWAS method to detect epistasis will be a key for discovering sophisticated pathogenesis, which is especially important for complex diseases such as Alzheimer's disease (AD).<h4>Results</h4>In this regard, this study presents GenEpi, a computational package to uncover epistasis associated with phenotypes by the proposed machine learning approach. GenEpi identifies both within-gene and cross-gene epistasis through a two-stage modeling workflow. In both stages, GenEpi adopts two-element combinatorial encoding when producing features and constructs the prediction models by L1-regularized regression with stability selection. The simulated data showed that GenEpi outperforms other widely-used methods on detecting the ground-truth epistasis. As real data is concerned, this study uses AD as an example to reveal the capability of GenEpi in finding disease-related variants and variant interactions that show both biological meanings and predictive power.<h4>Conclusions</h4>The results on simulation data and AD demonstrated that GenEpi has the ability to detect the epistasis associated with phenotypes effectively and efficiently. The released package can be generalized to largely facilitate the studies of many complex diseases in the near future.

<h4>Background</h4>Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown.<h4>Results</h4>We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells.<h4>Conclusions</h4>Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.

Single-cell RNA sequencing has emerged as a powerful tool for characterizing cells, but not all phenotypes of interest can be observed through changes in gene expression. Linking sequencing with optical analysis has provided insight into the molecular basis of cellular function, but current approaches have limited throughput. Here, we present a high-throughput platform for linked optical and gene expression profiling of single cells. We demonstrate accurate fluorescence and gene expression measurements on thousands of cells in a single experiment. We use the platform to characterize DNA and RNA changes through the cell cycle and correlate antibody fluorescence with gene expression. The platform's ability to isolate rare cell subsets and perform multiple measurements, including fluorescence and sequencing-based analysis, holds potential for scalable multi-modal single-cell analysis.

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from <i>Escherichia coli</i>, <i>Bacillus subtilis</i>, and <i>Staphylococcus aureus</i> in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in <i>S. aureus</i> ONC212 suppressed the proliferation of a number of Gram-positive (<i>S. aureus</i>, <i>B. subtilis</i>, and <i>Enterococcus faecium</i>) and Gram-negative species (<i>E. coli</i> and <i>Neisseria gonorrhoeae</i>). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant <i>S. aureus</i> persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.

Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

GABA_A receptor modulators are structurally almost as diverse as their target protein. A plethora of heterocyclic scaffolds has been described as modulating this extremely important receptor family. Some made it into clinical trials and, even on the market, some were dismissed. This review focuses on the synthetic accessibility and potential for library synthesis of GABA_A receptor modulators containing at least one heterocyclic scaffold, which were disclosed within the last 10 years.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sufficient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord differentiation.

New <i>Vinca</i> alkaloid derivatives were synthesized to improve the biological activity of the natural alkaloid vindoline. To this end, experiments were performed to link vindoline with various structural units, such as amino acids, a 1,2,3-triazole derivative, morpholine, piperazine and N-methylpiperazine. The structure of the new compounds was characterized by NMR spectroscopy and mass spectrometry (MS). Several compounds exhibited in vitro antiproliferative activity against human gynecological cancer cell lines with IC₅₀ values in the low micromolar concentration range.

Lecithin-encapsulated carvacrol has been incorporated into poly (vinyl alcohol) (PVA) for the purpose of obtaining active films for food packaging application. The influence of molecular weight (Mw) and degree of hydrolysis (DH) of the polymer on its ability to retain carvacrol has been analysed, as well as the changes in the film microstructure, thermal behaviour, and functional properties as packaging material provoked by liposome incorporation into PVA matrices. The films were obtained by casting the PVA aqueous solutions where liposomes were incorporated until reaching 0 (non-loaded liposomes), 5 or 10 g carvacrol per 100 g polymer. The non-acetylated, high Mw polymer provided films with a better mechanical performance, but less CA retention and a more heterogeneous structure. In contrast, partially acetylated, low Mw PVA gave rise to more homogenous films with a higher carvacrol content. Lecithin enhanced the thermal stability of both kinds of PVA, but reduced the crystallinity degree of non-acetylated PVA films, although it did not affect this parameter in acetylated PVA when liposomes contained carvacrol. The mechanical and barrier properties of the films were modified by liposome incorporation in line with the induced changes in crystallinity and microstructure of the films.

We repurposed the antifibrotic drug pirfenidone-which is approved for treatment of idiopathic lung fibrosis-in a series of patients with nonalcoholic steatohepatitis-related cirrhosis. Our report demonstrates the observed improvements in necroinflammation and regression of cirrhosis with pirfenidone use for 12-weeks, associated with classical hepatic repair complex features on follow-up liver biopsies. This novel work could help stimulate further randomized trials of pirfenidone in patients with nonalcoholic steatohepatitis-related liver fibrosis or cirrhosis, for whom no recommended drug treatments exists currently.

Perturbed neuronal migration and abnormal axonogenesis have been shown to be implicated in the pathogenesis of autism spectrum disorder (ASD). However, the molecular mechanism remains unknown. Here we demonstrate that dendritic cell factor 1(DCF1) is involved in neuronal migration and axonogenesis. The deletion of dcf1 in mice delays the localization of callosal projection neurons, while dcf1 overexpression restores normal migration. Delayed neurons appear as axon swelling and axonal boutons loss, resulting in a permanent deficit in the callosal projections. Western blot analysis indicates that absence of dcf1 leads to the abnormal activation of ERK signal. Differential protein expression assay shows that PEBP1, a negative regulator of the ERK signal, is significant downregulation in dcf1 KO mice. Direct interaction between DCF1 and PEBP1 is confirmed by Co-immunoprecipitation test, thus indicating that DCF1 regulates the ERK signal in a PEBP1-dependent pattern. As a result of the neurodevelopmental migration disorder, dcf1 deletion results in ASD-like behaviors in mice. This finding identifies a link between abnormal activated ERK signaling, delayed neuronal migration and autistic-like behaviors in humans.

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy-to-express antibody, whereas Erp27 and Erp57 co-overexpression increased cell density, viability, and the yield of difficult-to-express proteins. Foxa1 overexpression increased cell density, cell viability, and easy- and difficult-to-express protein yields, whereas it decreased reactive oxygen species late in fed-batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult- and/or easy-to-express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

Chemical investigation of secondary metabolites from the endophytic fungus <i>Pseudopestalotiopsis theae</i> led to the isolation of eighteen new polyketide derivatives, pestalotheols I-Q (<b>1</b>-<b>9</b>) and cytosporins O-W (<b>15</b>-<b>23</b>), together with eight known analogs (<b>10</b>-<b>14</b> and <b>24</b>-<b>26</b>). The structures of the new compounds were elucidated by HRMS and 1D and 2D NMR data, as well as by comparison with literature data. Modified Mosher's method was applied to determine the absolute configuration of some compounds. Compound <b>23</b> showed significant cytotoxicity against the mouse lymphoma cell line L5178Y with an IC₅₀ value of 3.0 μM. Furthermore, compounds <b>22</b> and <b>23</b> showed moderate antibacterial activity against drug-resistant <i>Acinetobacter baumannii</i> (ATCC BAA-1605) in combination with sublethal colistin concentrations.

Neurodegenerative diseases are a large group of neurological disorders with diverse etiological and pathological phenomena. However, current therapeutics rely mostly on symptomatic relief while failing to target the underlying disease pathobiology. G-protein-coupled receptors (GPCRs) are one of the most frequently targeted receptors for developing novel therapeutics for central nervous system (CNS) disorders. Many currently available antipsychotic therapeutics also act as either antagonists or agonists of different GPCRs. Therefore, GPCR-based drug development is spreading widely to regulate neurodegeneration and associated cognitive deficits through the modulation of canonical and noncanonical signals. Here, GPCRs' role in the pathophysiology of different neurodegenerative disease progressions and cognitive deficits has been highlighted, and an emphasis has been placed on the current pharmacological developments with GPCRs to provide an insight into a potential therapeutic target in the treatment of neurodegeneration.

Diethylstilbestrol (DES) is a synthetic estrogen and proven human teratogen and carcinogen reported to act via the estrogen receptor α (ERα). Since the endogenous ERα ligand 17β-estradiol (E2) does not show these adverse effects to a similar extent, we hypothesized that DES' interaction with the ERα differs from that of E2. The current study aimed to investigate possible differences between DES and E2 using in vitro assays that detect ERα-mediated effects, including ERα-mediated reporter gene expression, ERα-mediated breast cancer cell (T47D) proliferation and ERα-coregulator interactions and gene expression in T47D cells. Results obtained indicate that DES and E2 activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, significant differences between DES- and E2-induced binding of the ERα to 15 coregulator motifs and in transcriptomic signatures obtained in the T47D cells were observed. It is concluded that differences observed in binding of the ERα with several co-repressor motifs, in downregulation of genes involved in histone deacetylation and DNA methylation and in upregulation of CYP26A1 and CYP26B1 contribute to the differential effects reported for DES and E2.

<h4>Background</h4>Risky behaviors can lead to huge economic and health losses. However, limited efforts are paid to explore the genetic mechanisms of risky behaviors.<h4>Result</h4>MASH analysis identified a group of target genes for risky behaviors, such as APBB2, MAPT and DCC. For GO enrichment analysis, FUMA detected multiple risky behaviors related GO terms and brain related diseases, such as regulation of neuron differentiation (adjusted <i>P</i> value = 2.84×10^-5), autism spectrum disorder (adjusted <i>P</i> value =1.81×10^-27) and intelligence (adjusted <i>P</i> value =5.89×10^-15).<h4>Conclusion</h4>We reported multiple candidate genes and GO terms shared by the four risky behaviors, providing novel clues for understanding the genetic mechanism of risky behaviors.<h4>Methods</h4>Multivariate Adaptive Shrinkage (MASH) analysis was first applied to the GWAS data of four specific risky behaviors (automobile speeding, drinks per week, ever-smoker, number of sexual partners) to detect the common genetic variants shared by the four risky behaviors. Utilizing genomic functional annotation data of SNPs, the SNPs detected by MASH were then mapped to target genes. Finally, gene set enrichment analysis of the identified candidate genes were conducted by the FUMA platform to obtain risky behaviors related gene ontology (GO) terms as well as diseases and traits, respectively.

Acquired resistance and intrinsic to sorafenib therapy represents a major hurdle in improving the management of advanced hepatocellular carcinoma (HCC), which has been recently shown to be associated with the emergence of liver cancer stem cells (CSCs). However, it remains largely unknown whether and how histone posttranslational modifications, especially H3K27me3, are causally linked to the maintenance of self-renewal ability in sorafenib-resistant HCC. Here, we found that NOTCH1 signaling was activated in sorafenib-resistant HCC cells and NOTCH1 activation conferred hepatoma cells sorafenib resistance through enhanced self-renewal and tumorigenecity. Besides, the overexpression of EZH2 was required for the emergence of cancer stem cells following prolonged sorafenib treatment. As such, modulating EZH2 expression or activity suppressed activation of NOTCH1 pathway by elevating the expression of NOTCH1-related microRNAs, hsa-miR-21-5p and has-miR-26a-1-5p, via H3K27me3, and consequently weakened self-renewal ability and tumorigenecity and restored the anti-tumor effects of sorafenib. Overall, our results highlight the role of EZH2/NICD1 axis, and also suggest that EZH2 and NOTCH1 pathway are rational targets for therapeutic intervention in sorafenib-resistant HCC.

The extraordinary flexibility and structural heterogeneity of intrinsically disordered proteins (IDP) make them functionally versatile molecules. We have now begun to better understand their fundamental role in biology, however many aspects of their behaviour remain difficult to grasp experimentally. This is especially true for the intermolecular interactions which lead to the formation of transient or highly dynamic supramolecular self-assemblies, such as oligomers, aggregation intermediates and biomolecular condensates. Both the emerging functions and pathogenicity of these structures have stimulated great efforts to develop methodologies capable of providing useful insights. Significant progress in solution NMR spectroscopy has made this technique one of the most powerful to describe structural and dynamic features of IDPs within such assemblies at atomic resolution. Here, we review the most recent works that have illuminated key aspects of IDP assemblies and contributed significant advancements towards our understanding of the complex conformational landscape of prototypical disease-associated proteins. We also include a primer on some of the fundamental and innovative NMR methods being used in the discussed studies.

Peroxiredoxins (PRDXs) are members of a highly conserved peroxidase family and maintain intracellular reactive oxygen species (ROS) homeostasis. The family members are expressed in most organisms and involved in various biological processes, such as cellular protection against ROS, inflammation, carcinogenesis, atherosclerosis, heart diseases, and metabolism. In mammals, six PRDX members have been identified and are subdivided into three subfamilies: typical 2-Cys (PRDX1, PRDX2, PRDX3, and PRDX4), atypical 2-Cys (PRDX5), and 1-Cys (PRDX6) subfamilies. Knockout mouse models of PRDXs have been developed to investigate their in vivo roles. This review presents an overview of the knockout mouse models of PRDXs with emphases on the biological and physiological changes of these model mice.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and β2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-β2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-β2 inhibition. This study also highlights the first association between ADRβ2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRβ2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

Recently, adeno-associated virus (AAV)-mediated gene therapies have attracted clinical interest for treating neurodegenerative diseases including spinal muscular atrophy (SMA), Canavan disease (CD), Parkinson's disease (PD), and Friedreich's ataxia (FA). The influx of clinical findings led to the first approved gene therapy for neurodegenerative disorders in 2019 and highlighted new safety concerns for patients. Large doses of systemically administered AAV stimulate host immune responses, resulting in anti-capsid and anti-transgene immunity with implications for transgene expression, treatment longevity, and patient safety. Delivering lower doses directly to the central nervous system (CNS) is a promising alternative, resulting in higher transgene expression with decreased immune responses. However, neuroinflammatory responses after CNS-targeted delivery of AAV are a critical concern. Reported signs of AAV-associated neuroinflammation in preclinical studies include dorsal root ganglion (DRG) and spinal cord pathology with mononuclear cell infiltration. In this review, we discuss ways to manage neuroinflammation, including choice of AAV capsid serotypes, CNS-targeting routes of delivery, genetic modifications to the vector and/or transgene, and adding immunosuppressive strategies to clinical protocols. As additional gene therapies for neurodegenerative diseases enter clinics, tracking biomarkers of neuroinflammation will be important for understanding the impact immune reactions can have on treatment safety and efficacy.

A series of multi-target-directed ligands (MTDLs), obtained by attachment of a hydroxyphenylbenzimidazole (BIM) unit to donepezil (DNP) active mimetic moiety (benzyl-piperidine/-piperazine) was designed, synthesized, and evaluated as potential anti-Alzheimer's disease (AD) drugs in terms of biological activity (inhibition of acetylcholinesterase (AChE) and β-amyloid (Aβ) aggregation), metal chelation, and neuroprotection capacity. Among the DNP-BIM hybrids studied herein, the structural isomerization did not significantly improve the biological properties, while some substitutions, namely fluorine atom in each moiety or the methoxy group in the benzyl ring, evidenced higher cholinergic AChE activity. All the compounds are able to chelate Cu and Zn metal ions through their bidentate BIM moieties, but compound <b>5</b>, containing a three-dentate chelating unit, is the strongest Cu(II) chelator. Concerning the viability on neuroblastoma cells, compounds <b>9</b> and <b>10</b> displayed the highest reduction of Aβ-induced cell toxicity. In silico calculations of some pharmacokinetic descriptors indicate that all the compounds but the nitro derivatives have good potential oral-bioavailability. Overall, it can be concluded that most of the studied DNP-BIM conjugates showed quite good anti-AD properties, therefore deserving to be considered in further studies with the aim of understanding and treating AD.

A 68-year-old woman with chronic kidney disease receiving dialysis and iron supplementation presented to our hospital with painful blisters, fragile skin, and changes to skin pigmentation on the dorsal side of both upper and lower limbs. Skin biopsy findings and an increase in urine porphyrins confirmed the diagnosis of porphyria cutanea tarda. Upon examination, extremely high serum ferritin levels (6000 µg/L) suggested iron overload. Oral iron supplementation was immediately discontinued, and the patient received treatment with the iron chelators deferoxamine, 10 mg/kg/day intravenously for 4 days, and deferasirox, 540 mg/day orally. After a 4-month follow-up, ferritin levels were normal (97.7 µg/L) and the cutaneous manifestations of porphyria cutanea tarda had improved. Complete remission has been maintained for the last 2 years, and the patient's liver and heart function are normal. This case of porphyria cutanea tarda caused by secondary hemosiderosis highlights the potential toxicity of iron accumulation as a result of excessive iron supplementation. Although not approved for the treatment of patients on hemodialysis, we report the efficacy of deferasirox without any adverse effects in this case. We also stress the importance of the close monitoring of serum iron levels in kidney dialysis-and indeed all iron-supplemented-patients to avoid potential hepatic, cardiac, and endocrine damage.

<h4>Introduction</h4>Prospective memory (PM) is a marker of independent living in Alzheimer's disease. PM requires cue identification (prospective component) and remembering what should happen in response to the cue (retrospective component). We assessed neuroanatomical basis and functional relevance of PM.<h4>Methods</h4>84 older participants (53-94 years old, 58% male) with or without Mild Cognitive Impairment (MCI) performed PM tests, Activities of Daily Living (ADL) scale and had a structural MRI of the brain to estimate for cortical thickness and hippocampal subfield volumes. A General Linear Model cluster analysis was carried out using FreeSurfer to determine which cortical regions were correlated with PM scores.<h4>Results</h4>Both components of PM are impaired in MCI (p < .001). The retrospective component of PM correlates strongly with ADL (p = .005). Prospective component performance correlates positively with cortical thickness of bilateral frontal-temporal-parietal cortex and volume of CA1 of hippocampus. In contrast, the retrospective component performance correlates positively with cortical thickness of a right-lateralised fronto-temporal-parietal network and volumes of subiculum and CA3 hippocampal subfields.<h4>Discussion</h4>Our neuroimaging findings complement and extend previous research into structural correlates of PM. Here, we show that there are distinct, yet, overlapping brain regions correlating with the two components of PM. PM performance provides a window into real-life functional abilities in people at risk of Alzheimer's disease and could be utilised as a marker of clinically relevant disease.

Social affiliative behaviors-engagement in positive (i.e., nonaggressive) social approach and reciprocal social interactions with a conspecific-comprise a construct within the National Institute of Mental Health Research Domain Criteria Social Processes Domain. These behaviors are disrupted in multiple human neurodevelopmental and neuropsychiatric disorders, such as autism, schizophrenia, social phobia, and others. Human genetic studies have strongly implicated synaptic cell adhesion molecules (sCAMs) in several such disorders that involve marked reductions, or other dysregulations, of social affiliative behaviors. Here, we review the literature on the role of sCAMs in social affiliative behaviors. We integrate findings pertaining to synapse structure and morphology, neurotransmission, postsynaptic signaling pathways, and neural circuitry to propose a multilevel model that addresses the impact of a diverse group of sCAMs, including neurexins, neuroligins, protocadherins, immunoglobulin superfamily proteins, and leucine-rich repeat proteins, as well as their associated scaffolding proteins, including SHANKs and others, on social affiliative behaviors. This review finds that the disruption of sCAMs often manifests in changes in social affiliative behaviors, likely through alterations in synaptic maturity, pruning, and specificity, leading to excitation/inhibition imbalance in several key regions, namely the medial prefrontal cortex, basolateral amygdala, hippocampus, anterior cingulate cortex, and ventral tegmental area. Unraveling the complex network of interacting sCAMs in glutamatergic synapses will be an important strategy for elucidating the mechanisms of social affiliative behaviors and the alteration of these behaviors in many neuropsychiatric and neurodevelopmental disorders.

Supramolecular capsules can be used to change the activity and selectivity of a catalyst through the influence of the second coordination sphere, reminiscent of how enzymes control the selectivity of their processes. In enzymes, this approach is used to also control the enantioselectivity of reactions in which the active catalytic site is often not chiral but the second coordination sphere is. We are interested in the possibility to generate a chiral second coordination sphere around an otherwise achiral transition metal complex for asymmetric catalysis. In this paper we show that the ligand template approach can be used to generate a chiral second coordination sphere around a rhodium complex, which is used in asymmetric hydroformylation.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity <i>in vitro</i> and <i>in vivo</i>. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

Medicinal chemistry plays a fundamental and underlying role in chemical biology, pharmacology, and medicine to discover safe and efficacious drugs. Small molecule medicinal chemistry relies on iterative learning cycles composed of compound design, synthesis, testing, and data analysis to provide new chemical probes and lead compounds for novel and druggable targets. Using traditional approaches, the time from hypothesis to obtaining the results can be protracted, thus limiting the number of compounds that can be advanced into clinical studies. This challenge can be tackled with the recourse of enabling technologies that are showing great potential in improving the drug discovery process. In this Perspective, we highlight recent developments toward innovative medicinal chemistry strategies based on continuous flow systems coupled with automation and bioassays. After a discussion of the aims and concepts, we describe equipment and representative examples of automated flow systems and end-to-end prototypes realized to expedite medicinal chemistry discovery cycles.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by increased uptake and accumulation of lipids in hepatocytes. Simple steatosis may progress to non-alcoholic steatohepatitis (NASH) with inflammation, hepatocellular injury and fibrosis. CCN1 is an important matrix protein that regulates cell death and promotes immune cell adhesion and may potentially control this process. The role of CCN1 in NASH remains unclear. We investigated the role of CCN1 in the pathogenesis of steatohepatitis. CCN1 upregulation was found to be closely related with steatosis in patients with NASH, obese mice and a FFA-treated hepatocyte model. Controlling the expression of CCN1 in murine NASH models demonstrated that CCN1 increased the severity of steatosis and inflammation. From the sequence results, we found that fatty acid metabolism genes were primarily involved in the MCD mice overexpressing CCN1 compared to the control. Then, the expression of fatty acid metabolism genes was determined using a custom-designed pathway-focused qPCR-based gene expression array. Expression analysis showed that CCN1 overexpression significantly upregulated the expression of fatty acid metabolism-associated genes. In vitro analysis revealed that CCN1 increased the intracellular TG content, the pro-inflammatory cytokines and the expression level of apoptosis-associated proteins in a steatosis model using murine primary hepatocytes. We identified CCN1 as an important positive regulator in NASH.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.

Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUG^exp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse <i>HSA</i>^LR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUG^exp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.

<h4>Objective</h4>To assess the value of qualitative and quantitative MRI radiomics features for noninvasive prediction of immuno-oncologic characteristics and outcomes of hepatocellular carcinoma (HCC).<h4>Methods</h4>This retrospective, IRB-approved study included 48 patients with HCC (M/F 35/13, mean age 60y) who underwent hepatic resection or transplant within 4 months of abdominal MRI. Qualitative imaging traits, quantitative nontexture related and texture features were assessed in index lesions on contrast-enhanced T1-weighted and diffusion-weighted images. The association of imaging features with immunoprofiling and genomics features was assessed using binary logistic regression and correlation analyses. Binary logistic regression analysis was also employed to analyse the association of radiomics, histopathologic and genomics features with radiological early recurrence of HCC at 12 months.<h4>Results</h4>Qualitative (r = - 0.41-0.40, p < 0.042) and quantitative (r = - 0.52-0.45, p < 0.049) radiomics features correlated with immunohistochemical cell type markers for T-cells (CD3), macrophages (CD68) and endothelial cells (CD31). Radiomics features also correlated with expression of immunotherapy targets PD-L1 at protein level (r = 0.41-0.47, p < 0.029) as well as PD1 and CTLA4 at mRNA expression level (r = - 0.48-0.47, p < 0.037). Finally, radiomics features, including tumour size, showed significant diagnostic performance for assessment of early HCC recurrence (AUC 0.76-0.80, p < 0.043), while immunoprofiling and genomic features did not (p = 0.098-0929).<h4>Conclusions</h4>MRI radiomics features may serve as noninvasive predictors of HCC immuno-oncological characteristics and tumour recurrence and may aid in treatment stratification of HCC patients. These results need prospective validation.<h4>Key points</h4>• MRI radiomics features showed significant associations with immunophenotyping and genomics characteristics of hepatocellular carcinoma. • Radiomics features, including tumour size, showed significant associations with early hepatocellular carcinoma recurrence after resection.

<h4>Background</h4>Real-time bedside information on regional ventilation and perfusion during mechanical ventilation (MV) may help to elucidate the physiological and pathophysiological effects of MV settings in healthy and injured lungs. We aimed to study the effects of positive end-expiratory pressure (PEEP) and tidal volume (V_T) on the distributions of regional ventilation and perfusion by electrical impedance tomography (EIT) in healthy and injured lungs.<h4>Methods</h4>One-hit acute lung injury model was established in 6 piglets by repeated lung lavages (injured group). Four ventilated piglets served as the control group. A randomized sequence of any possible combination of three V_T (7, 10, and 15 ml/kg) and four levels of PEEP (5, 8, 10, and 12 cmH₂O) was performed in all animals. Ventilation and perfusion distributions were computed by EIT within three regions-of-interest (ROIs): nondependent, middle, dependent. A mixed design with one between-subjects factor (group: intervention or control), and two within-subjects factors (PEEP and V_T) was used, with a three-way mixed analysis of variance (ANOVA).<h4>Results</h4>Two-way interactions between PEEP and group, and V_T and group, were observed for the dependent ROI (p = 0.035 and 0.012, respectively), indicating that the increase in the dependent ROI ventilation was greater at higher PEEP and V_T in the injured group than in the control group. A two-way interaction between PEEP and V_T was observed for perfusion distribution in each ROI: nondependent (p = 0.030), middle (p = 0.006), and dependent (p = 0.001); no interaction was observed between injured and control groups.<h4>Conclusions</h4>Large PEEP and V_T levels were associated with greater pulmonary ventilation of the dependent lung region in experimental lung injury, whereas they affected pulmonary perfusion of all lung regions both in the control and in the experimental lung injury groups.

<h4>Background</h4>Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Skin barrier defects contribute to disease initiation and development; however, underlying mechanisms remain elusive.<h4>Objective</h4>To understand the underlying cause of barrier defect, we investigated aberrant expression of specific microRNAs (miRNAs) in AD. Delineating the molecular mechanism of dysregulated miRNA network, we focused on identification of specific drugs that can modulate miRNA expression and repair the defective barrier in AD.<h4>Methods</h4>A screen for differentially expressed miRNAs between healthy skin and AD lesional skin resulted in the identification of miR-335 as the most consistently downregulated miRNA in AD. Using in silico prediction combined with experimental validation, we characterized downstream miR-335 targets and elucidated the molecular pathways by which this microRNA maintains epidermal homeostasis in healthy skin.<h4>Results</h4>miR-335 was identified as a potent inducer of keratinocyte differentiation; it exerts this effect by directly repressing SOX6. By recruiting SMARCA complex components, SOX6 suppresses epidermal differentiation and epigenetically silences critical genes involved in keratinocyte differentiation. In AD lesional skin, miR-335 expression is aberrantly lost. SOX6 is abnormally expressed throughout the epidermis, where it impairs skin barrier development. We demonstrate that miR-335 is epigenetically regulated by histone deacetylases; a screen for suitable histone deacetylase inhibitors identified belinostat as a candidate drug that can restore epidermal miR-335 expression and rescue the defective skin barrier in AD.<h4>Conclusion</h4>Belinostat is of clinical significance not only as a candidate drug for AD treatment, but also as a potential means of stopping the atopic march and further progression of this systemic allergic disease.

Based on a live attenuated <i>Yersinia pestis</i> KIM10(pCD1Ap) strain (Pgm^-, pPCP1^-), we attempted to engineer its lipid A species to achieve improvement of immunogenicity and safety. A mutant strain designated as YPS19(pCD1Ap), mainly synthesizing the hexa-acylated lipid A, and another mutant strain designated as YPS20(pCD1Ap), synthesizing 1-dephosphalated hexa-acylated lipid A (detoxified lipid A), presented relatively low virulence in comparison to KIM10(pCD1Ap) by intramuscular (i.m.) or subcutaneous (s.c.) administration. The i.m. administration with either the KIM10(pCD1Ap) or YPS19(pCD1Ap) strain afforded significant protection against bubonic and pneumonic plague compared to the s.c. administration, while administration with completely attenuated YPS20(pCD1Ap) strain failed to afford significant protection. Antibody analysis showed that i.m. administration induced balanced Th1 and Th2 responses but s.c. administration stimulated Th2-biased responses. Safety evaluation showed that YPS19(pCD1Ap) was relatively safer than its parent KIM10(pCD1Ap) in Hfe^-/- mice manifesting iron overload in tissues, which also did not impair its protection. Therefore, the immune activity of hexa-acylated lipid A can be harnessed for rationally designing bacteria-derived vaccines.

In the biological process of testicular spermatogenesis, the expression and interaction of many genes are regulated by microRNAs (miRNAs). However, comparisons of miRNA expression between descended testes (DTs) and undescended testes (UDTs) are rarely done in horses. In this study, we selected two UDTs (CKY2b and GU4b) from Chakouyi (CKY) and Guanzhong (GU) horses and eight DTs (GU1-3, CKY1, CKY3, CKY2a, GU4a, and GU5). Three groups were compared to evaluate expression patterns of testicular miRNA in stallion testes. Group 1 compared normal CKY horses and GU horses (CKY1 and CKY3 vs. GU1-3). Group 2 (CKY2a and GU4a (DTs) vs. CKY2b and GU4b (UDTs)) and group 3 (GU1-3, CKY1, CKY3 (DTs) vs. CKY2b and GU4b (UDTs)) compared the expression levels in unilateral retained testes to normal testes. The results show that 42 miRNAs (7 upregulated and 35 downregulated) had significantly different expression levels in both comparisons. The expression levels of eca-miR-545, eca-miR-9084, eca-miR-449a, eca-miR-9024, eca-miR-9121, eca-miR-8908e, eca-miR-136, eca-miR-329b, eca-miR-370, and eca-miR-181b were further confirmed by quantitative real-time PCR assay. The target genes of differentially expressed miRNAs in three comparisons were predicted, and the functions were annotated. The putative target genes of the 42 co-differentially expressed miRNAs were annotated to 15 functional terms, including metal ion binding, GTPase activator activity, zinc ion binding, intracellular, cytoplasm, and cancer pathways, and osteoclast differentiation. Our data indicate that the differentially expressed miRNAs in undescended testis suggests a potential role in male fertility and a relationship with cryptorchidism in horses. The discovery of miRNAs in stallion testes might contribute to a new direction in the search for biomarkers of stallion fertility.

Mesenchymal stem/stromal cells (MSCs) are a reservoir for tissue homeostasis and repair that age during organismal aging. Beside the fundamental in vivo role of MSCs, they have also emerged in the last years as extremely promising therapeutic agents for a wide variety of clinical conditions. MSC use frequently requires in vitro expansion, thus exposing cells to replicative senescence. Aging of MSCs (both in vivo and in vitro) can affect not only their replicative potential, but also their properties, like immunomodulation and secretory profile, thus possibly compromising their therapeutic effect. It is therefore of critical importance to unveil the underlying mechanisms of MSC senescence and to define shared methods to assess MSC aging status. The present review will focus on current scientific knowledge about MSC aging mechanisms, control and effects, including possible anti-aging treatments.

Artichoke by-products represent a high amount of waste whose removal entails several costs. Moreover, feed is the main cost in a farm. So, including these by-products in ruminant diets would lower feed costs. Two experiments were conducted to evaluate the effect of two levels of inclusion, 12.5% and 25.0%, of two silages of artichoke by-products (artichoke bracts, AB and artichoke plant, AP) in the diet of goats on the milk yield, composition and quality and on the metabolic profile of the animals. AB presented the lowest blood urea content and there were no differences in milk yield in the two experiments. However, with 25.0% of silage by-product in the diet, a higher fat content was observed in AB and of protein in AP, as well as this treatment showing a slightly higher Se content. Regarding the milk lipid profile, milk from 12.5% of AP treatment presented a higher PUFA content. In conclusion, the use of silage artichoke by-products in dairy goat diets does not jeopardise milk yield and quality and health status of animals and, from a nutritional point of view for human health, a slightly better mineral and lipid profile is observed in milk from AP treatments.

In the current study, we hypothesized that extracellular vesicles (EVs) secreted from human papilloma virus (HPV)-infected cervical cancer cells exacerbate human immunodeficiency virus (HIV)-1 replication in differentiated U1 cell line through an oxidative stress pathway. To test the hypothesis, we treated an HIV-1-infected macrophage cell line (U1) with HPV-infected Caski cell culture supernatant (CCS). We observed a significant increase in HIV-1 replication, which was associated with an increase in the expression of cytochrome P450 (CYPs 1A1 and 2A6) in the CCS-treated U1 cells. Furthermore, we isolated EVs from CCS (CCS-EVs), which showed the presence of CYPs (1A1, 2A6), superoxide dismutase 1 (SOD1), and HPV oncoproteins HPV16 E6. CCS-EVs when exposed to the U1 cells also significantly increased HIV-1 replication. Treatment of antioxidant, CYP1A1 and CYP2A6 inhibitors, and chemodietary agents with antioxidant properties significantly reduced the CCS and CCS-EVs mediated HIV-1 replication in U1 cells. Altogether, we demonstrate that cervical cancer cells exacerbate HIV-1 replication in differentiated U1 cell line via transferring CYPs and HPV oncoproteins through EVs. We also show that the viral replication occurs via CYP and oxidative stress pathways, and the viral replication is also reduced by chemodietary agents. This study provides important information regarding biological interactions between HPV and HIV-1 via EVs leading to enhanced HIV-1 replication.

Embryonic stem cells (ESCs) and adult stem cells (ASCs) possess the remarkable capacity to self-renew while remaining poised to differentiate into multiple progenies in the context of a rapidly developing embryo or in steady-state tissues, respectively. This ability is controlled by complex genetic programs, which are dynamically orchestrated at different steps of gene expression, including chromatin remodeling, mRNA transcription, processing, and stability. In addition to maintaining stem cell homeostasis, these molecular processes need to be rapidly rewired to coordinate complex physiological modifications required to redirect cell fate in response to environmental clues, such as differentiation signals or tissue injuries. Although chromatin remodeling and mRNA expression have been extensively studied in stem cells, accumulating evidence suggests that stem cell transcriptomes and proteomes are poorly correlated and that stem cell properties require finely tuned protein synthesis. In addition, many studies have shown that the biogenesis of the translation machinery, the ribosome, is decisive for sustaining ESC and ASC properties. Therefore, these observations emphasize the importance of translational control in stem cell homeostasis and fate decisions. In this review, we will provide the most recent literature describing how ribosome biogenesis and translational control regulate stem cell functions and are crucial for accommodating proteome remodeling in response to changes in stem cell fate.

Human cells express large amounts of different proteins continuously that must fold into well-defined structures that need to remain correctly folded and assemble in order to ensure their cellular and biological functions. The integrity of this protein balance/homeostasis, also named proteostasis, is maintained by the proteostasis network (PN). This integrated biological system, which comprises about 2000 proteins (chaperones, folding enzymes, degradation components), control and coordinate protein synthesis folding and localization, conformational maintenance, and degradation. This network is particularly challenged by mutations such as those found in genetic diseases, because of the inability of an altered peptide sequence to properly engage PN components that trigger misfolding and loss of function. Thus, deletions found in the ΔF508 variant of the Cystic Fibrosis (CF) transmembrane regulator (CFTR) triggering CF or missense mutations found in the Z variant of Alpha 1-Antitrypsin deficiency (AATD), leading to lung and liver diseases, can accelerate misfolding and/or generate aggregates. Conversely to CF variants, for which three correctors are already approved (ivacaftor, lumacaftor/ivacaftor, and most recently tezacaftor/ivacaftor), there are limited therapeutic options for AATD. Therefore, a more detailed understanding of the PN components governing AAT variant biogenesis and their manipulation by pharmacological intervention could delay, or even better, avoid the onset of AATD-related pathologies.

<h4>Introduction</h4>Curcumin (Cur) is a natural extract of Asian spice <i>Curcumin longa</i>, showing multi-targeting capability and low toxicity in anti-tumor activities. The low bioavailability restricts its application as a therapeutic agent. Folate (FA) receptors are highly expressed in many malignant tumors while low expressed in normal tissue. Herein, we developed a self-assembled FA modified MPEG-PCL micelle to incorporate Cur (FA/Nano-Cur) and applied it for colorectal cancer therapy.<h4>Methods</h4>We prepared FA/Nano-Cur micelles and identified their characteristics. The drug release behavior, pharmacokinetics and in vitro anti-tumor activities of FA/Nano-Cur were studied. Furthermore, the in vivo anti-tumor ability assessment and anti-tumor mechanisms investigation were carried out in murine colorectal cancer model.<h4>Results</h4>FA/Nano-Cur micelles had an average particle size of 30.47 nm. Elongated T_1/2 and larger AUC were found in FA/Nano-Cur group than that in the Free Cur group. MTT assay and apoptotic study indicated the growth inhibitory effect and pro-apoptotic effect of FA/Nano-Cur were the most significant among all treatments. Moreover, the in vivo study demonstrated that FA/Nano-Cur micelles exhibited a much stronger effect to suppress tumor growth, promote tumor apoptosis and attenuate tumor angiogenesis than Free Cur and Nano-Cur micelles.<h4>Conclusion</h4>The present study demonstrated FA/Nano-Cur micelles might be a promising therapeutic agent in colorectal cancer treatment with distinctive advantages of improved bioavailability, sustained drug release, tumor-targeted delivery and low toxicity.

<h4>Background</h4>Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia.<h4>Objetive</h4>To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs).<h4>Methods</h4>We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels.<h4>Findings</h4>We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation.<h4>Main conclusions</h4>Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

<b>Context:</b> The hypothalamic circuit has an essential role in the regulation of appetite and energy expenditure. Pathogenic variants in genes involved in the hypothalamic leptin-melanocortin pathway, including melanocortin-4-receptor (<i>MC4R</i>), have been associated with monogenic obesity. <b>Objective:</b> To determine the rate and spectrum of rare variants in genes involved in melanocortin pathway or hypothalamic development in patients with severe early-onset obesity (height-adjusted weight >60% before age 10 years). <b>Methods:</b> We used a custom-made targeted exome sequencing panel to assess peripheral blood DNA samples for rare (minor allele frequency <0.5%), pathogenic/likely pathogenic variants in 24 genes related to the hypothalamic circuit in 92 subjects (51% males, median age 13.7 years) with early-onset severe obesity (median body mass index (BMI) Z-score + 4.0). <b>Results:</b> We identified a novel frameshift deletion in <i>MC4R</i> (p.V103Afs5^*) in two unrelated patients and a previously reported <i>MC4R</i> variant (p.T112M) in one patient. In addition, we identified rare heterozygous missense variants in <i>ADCY3</i> (p.G1110R), <i>MYT1L</i> (p.R807Q), <i>ISL1</i> (p.I347F), <i>LRP2</i> (p.R2479I, and p.N3315S) and a hemizygous missense variant in <i>GRPR</i> (p.L87M) (each in one patient), possibly contributing to the obesity phenotype in these patients. Altogether 8 % (7/92) of the subjects had rare pathogenic/likely pathogenic variants in the studied genes. <b>Conclusions:</b> Rare genetic variants within the hypothalamic circuit are prevalent and contribute to the development of severe early-onset obesity. Targeted exome sequencing is useful in identifying affected subjects. Further studies are needed to evaluate the variants' clinical significance and to define optimal treatment.

Long noncoding RNAs (lncRNAs) play important roles in the regulation of gene expression by acting as competing endogenous RNAs (ceRNAs). However, the roles of lncRNA-associated ceRNAs in oncogenesis are not fully understood. The present study aims to determine whether a ceRNA network can serve as a prognostic marker in human prostate cancer (PCa). In order to identify a ceRNA network and the key lncRNAs in PCa, we constructed a differentially expressed lncRNAs (DELs)-differentially expressed miRNAs (DEMis)-differentially expressed mRNAs (DEMs) regulatory network based on the ceRNA theory using data from the Cancer Genome Atlas (TCGA). We found that the DELs-DEMis-DEMs network was composed of 27 DELs nodes, seven DEMis nodes, and three DEMs nodes. The 27 DELs were further analyzed with several public databases to provide meaningful information for understanding the functional roles of lncRNAs in regulatory networks in PCa. We selected ADAMTS9-AS1 to determine its role in PCa and found that ADAMTS9-AS1 significantly influences tumor cell growth and proliferation, suggesting that it plays a tumor suppressive role. In addition, ADAMTS9-AS1 functioned as ceRNA, effectively becoming a sponge for hsa-mir-96 and modulating the expression of PRDM16. These results suggest that ceRNAs could accelerate biomarker discovery and therapeutic strategies for PCa.

<i>HFE</i>-related Hemochromatosis is the most common genetic iron overload disease in European populations, particularly of Nordic or Celtic ancestry. It is reported that the <i>HFE</i> p.C282Y mutation is present in 1/10 people of northern European descent, resulting in one in two hundred people will be homozygous. However, the <i>HFE</i> p.C282Y heterozygosity is virtually absent among East Asians, including Japanese, Koreans, and Chinese. In this article, we report a case of <i>HFE</i>-related hemochromatosis caused by compound heterozygosity <i>HFE</i> p.C282Y/p.R71X. This is the first report of hemochromatosis associated with <i>HFE</i> p.C282Y mutation in China.

Chemosensitization is an effective strategy to overcome the drawbacks of arsenic trioxide (As₂O₃) treatment, which may be possible through the use of dietary supplements in combination. The present investigation evaluates the synergistic mechanism of action of vitamins, such as L-ascorbic acid (L-AA) and α-tocopherol (α-TOC) in As₂O₃ chemotherapy using human leukemia (HL-60) cells. <i>In vitro</i> assays on the cytotoxicity of As₂O₃ and vitamins and cellular apoptotic evidences were done; a proteomic investigation with mass spectrometry was also performed. The combination of L-AA and α-TOC potentiates As₂O₃ cytotoxicity in HL-60 cells, substantiated by depletion in antioxidant status, mitochondrial transmembrane potential, and inhibition of nuclear factor erythroid 2-related factor 2 and B-cell lymphoma 2 transcription factors. Mass spectrometry results showed decreased expression of proteins regulating cell cycle and translation in cells treated with As₂O₃, L-AA, and α-TOC when compared with As₂O₃-treated sample. In addition, this combination treatment identified numerous proteins associated with apoptosis and cell stress. HL-60 cells became more prone to As₂O₃ on exposure to L-AA and α-TOC, indicating that this combination may be a promising approach to increase the outcome of As₂O₃ chemotherapy.

The endoplasmic reticulum (ER) is a dynamic membrane system comprising different and interconnected subdomains. The ER structure changes in response to different stress conditions through the activation of a selective autophagic pathway called ER-phagy. This represents a quality control mechanism for ER turnover and component recycling. Several ER-resident proteins have been indicated as receptors for ER-phagy; among these, there are proteins characterized by the presence of a reticulon homology domain (RHD). RHD-containing proteins promote ER fragmentation by a mechanism that involves LC3 binding and lysosome delivery. Moreover, the presence of a correct RHD structure is closely related to their capability to regulate ER shape and morphology by curvature induction and membrane remodeling. Deregulation of the ER-selective autophagic pathway due to defects in proteins with RHD has been implicated in several human diseases, infectious and neurodegenerative diseases in particular, as well as in cancer development. While the molecular mechanisms and the physiological role of ER-phagy are not yet fully understood, it is quite clear that this process is involved in different cellular signaling pathways and has an impact in several human pathologies.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the HTT gene. HD patient-specific induced pluripotent stem cells (iPSCs) represent an excellent model for the disease study. We generated iPSC line from blood mononuclear cells of HD patient with 38 CAG repeats in the HTT exon 1 using integration free episomal plasmids expressing Yamanaka factors. The iPSC line retained the disease causing mutation and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

Solid Recovered Fuels (SRF) include non-combustible mineral components (e.g. CaCO₃, SiO₂, Al₂O₃) that are required as raw materials for producing clinker and are completely incorporated into the clinker during the thermal recovery of SRF. This paper discusses simple and practicable ways of finding the relative amount of SRF that may be utilised as raw material (given as the recycling index). For this purpose, the entire mineral content of SRF was determined as the ash content and its main components were identified using different analytical methods.•A fusion melt of the previously incinerated sample with subsequent measuring using ICP-OES and XRF as well as a total digestion of the incinerated and non-incinerated sample with subsequent measuring using ICP-OES/ICP-MS were applied.•The results showed a good agreement of all four analytical methods for the elementary oxides Al₂O₃, CaO, Fe₂O₃, SiO₂, TiO₂, P₂O₅ and MgO (relative deviation from 6.6 to 38.9%) and slightly higher deviations for K₂O, Na₂O and SO₃ (14.2-96.0%).•It was also shown that different incineration temperatures (550 °C, 815 °C and 950 °C) have no effect on the result of the recycling index unless it is assumed that the recycling index equals the ash content.

The discovery of the CRISPR systems has enriched the application of gene therapy and biotechnology. As a type of robust and simple toolbox, the CRISPR system has greatly promoted the development of cellular signal sensors at the genomic level. Although CRISPR systems have demonstrated that they can be used in eukaryotic and even mammalian cells after extraction from prokaryotic cells, controlling their gene-editing activity remains a challenge. Here we summarize the advantages and disadvantages of building a CRIRPR-based signal sensor through sgRNA reconstruction, as well as possible ways to reprogram the signal network of cells. We also propose how to further improve the design of the current signal sensors based on sgRNA-riboswitch. We believe that the development of these technologies and the construction of platforms can further promote the development of environment detection, disease diagnosis, and gene therapy by means of synthetic biology.

Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) has been reported in cancers but its role and potential molecular mechanism in hepatocellular carcinoma (HCC) is unclear. Therefore, we aimed to investigate the clinical value and molecular mechanism of CELSR3 in HCC using an <i>in vitro</i> experiment, a meta-analysis and bioinformatics. The <i>in vitro</i> experiment determined the promoting effect of CELSR3 in the proliferation, invasion, and migration of HCC cells. CELSR3 knockout causes S-phage arrest in HCC cells. CELSR3 can also inhibit the apoptosis of HCC cells. The expression of the CELSR3 gene and protein was significantly elevated in HCC. Elevated CELSR3 was correlated to the bigger tumor size, higher pathological stage, and the worse overall survival of HCC. Methylation analysis revealed that the hypomethylation of CELSR3 regulated by DNMT1, DNMT3A, and DNMT3B may be the underlying mechanism of upregulated CELSR3. Biological enrichment analysis uncovered that the cell cycle, DNA replication, and PI3K-Akt signaling pathways were important pathways regulated by CELSR3 and its co-expressed genes in HCC. Taken together, upregulated CELSR3 is an important regulator in the progression and prognosis of HCC. The hypomethylation of CELSR3 and its regulation in the cell cycle may be the potential molecular mechanism in HCC.

The ubiquitin-proteasome system (UPS) governs the protein degradation process and balances proteostasis and cellular homeostasis. It is a well-controlled mechanism, in which removal of the damaged or excessive proteins is essential in driving signal pathways for cell survival or death. Accumulation of damaged proteins and failure in removal may contribute to disease initiation such as in cancers and neurodegenerative diseases. In this notion, specific protein-protein interaction is essential for the recognition of targeted proteins in UPS. WW domain plays an indispensable role in the protein-protein interactions during signaling. Among the 51 WW domain-containing proteins in the human proteomics, near one-quarter of them are involved in the UPS, suggesting that WW domains are crucial modules for driving the protein-protein binding and subsequent ubiquitination and degradation. In this review, we detail a broad spectrum of WW domains in protein-protein recognition, signal transduction, and relevance to diseases. New perspectives in dissecting the molecular interactions are provided.

A number of recent studies have highlighted the causes of bone nonunion (BN), however, the rate of BN incidence continues to rise and available therapeutic options to treat this condition remain limited. Thus, to prevent disease progression and improve patient prognosis, it is vital that BN, or the risk thereof, be accurately identified in a timely manner. In the present study, bioinformatics analyses were used to screen for the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) between patients with BN and those with bone union, using data from the Gene Expression Omnibus database. Furthermore, clinical samples were collected and analyzed by reverse transcription‑quantitative PCR and western blotting. In vitro and in vivo experiments were carried out to confirm the relationship between BN and the DEGs of interest, in addition to being used to explore the underlying molecular mechanism of BN. Functional enrichment analysis of the downregulated DEGs revealed them to be enriched for genes associated with 'ECM‑receptor interactions', 'focal adhesion', 'and the calcium signaling pathway'. When comparing DEM target genes with these DEGs, nine DEGs were identified as putative DEM targets, where hsa‑microRNA (miR)‑1225‑5p‑CCNL2, hsa‑miR‑339‑5p‑PRCP, and hsa‑miR‑193a‑3p‑mitogen‑activated protein kinase 10 (MAPK10) were the only three pairs which were associated with decreased gene expression levels. Furthermore, hsa‑miR‑193a‑3p was demonstrated to induce BN by targeting MAPK10. Collectively, the results of the present study suggest that hsa‑miR‑193a‑3p may be a viable biomarker of BN.

In December 2019, a novel coronavirus (SARS-CoV-2) was identified in patients with pneumonia (called COVID-19) in Wuhan, Hubei Province, China. SARS-CoV-2 shares high sequence similarity and uses the same cell entry receptor, angiotensin-converting enzyme 2 (ACE2), as does severe acute respiratory syndrome coronavirus (SARS-CoV). Several studies have provided bioinformatic evidence of potential routes for SARS-CoV-2 infection in respiratory, cardiovascular, digestive and urinary systems. However, whether the reproductive system is a potential target of SARS-CoV-2 infection has not been determined. Here, we investigate the expression pattern of ACE2 in adult human testis at the level of single-cell transcriptomes. The results indicate that ACE2 is predominantly enriched in spermatogonia, Leydig and Sertoli cells. Gene ontology analyses indicate that GO categories associated with viral reproduction and transmission are highly enriched in ACE2-positive spermatogonia while male gamete generation related terms are down-regulated. Cell-cell junction and immunity related GO terms are increased in ACE2-positive Leydig and Sertoli cells, but mitochondria and reproduction related GO terms are decreased. These findings provide evidence that human testes are a potential target of SARS-CoV-2 infection which may have significant impact on our understanding of the pathophysiology of this rapidly spreading disease.

<h4>Background: </h4> Genome-wide association studies have identified multiple genomic loci associated with coronary artery disease, but most are common variants in non-coding regions that provide limited information on causal genes and etiology of the disease. To better understand etiological pathways that might lead to discovery of new treatments or prevention strategies, we focused our investigation on low-frequency and rare sequence variations primarily residing in coding regions of the genome while also exploring associations with common variants. Methods and Results Using samples of individuals of European ancestry from ten cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, both cross-sectional and prospective analyses were conducted to examine associations between genetic variants and myocardial infarction (MI), coronary heart disease (CHD), and all-cause mortality following these events. Single variant and gene-based analyses were performed separately in each cohort and then meta-analyzed for each outcome. A low-frequency intronic variant (rs988583) in PLCL1 was significantly associated with prevalent MI (OR=1.80, 95% confidence interval: 1.43, 2.27; P =7.12 × 10 -7 ). Three common variants, rs9349379 in PHACTR1 , and rs1333048 and rs4977574 in the 9p21 region, were significantly associated with prevalent CHD. Four common variants (rs4977574, rs10757278, rs1333049, and rs1333048) within the 9p21 locus were significantly associated with incident MI. We conducted gene-based burden tests for genes with a cumulative minor allele count (cMAC) 5 and variants with minor allele frequency (MAF) < 5%. TMPRSS5 and LDLRAD1 were significantly associated with prevalent MI and CHD, respectively, and RC3H2 and ANGPTL4 were significantly associated with incident MI and CHD, respectively. No loci were significantly associated with all-cause mortality following a MI or CHD event. Conclusion This study confirmed previously reported loci influencing heart disease risk, and one single variant and three genes associated with MI and CHD were newly identified and warrant future investigation.

<h4>Background</h4>Prospective studies have suggested higher factor VIII (FVIII) levels are an independent risk factor for coronary heart disease (CHD) and stroke. However, limited information, including on genetic and epigenetic contributors to FVIII variation, is available specifically among African Americans (AAs), who have higher FVIII levels than Europeans.<h4>Objectives</h4>We measured FVIII levels in ~3400 AAs from the community-based Jackson Heart Study and assessed genetic, epigenetic, and epidemiological correlates of FVIII, as well as incident cardiovascular disease (CVD) associations.<h4>Methods</h4>We assessed cross-sectional associations of FVIII with CVD risk factors as well as incident CHD, stroke, heart failure, and mortality associations. We additionally assessed associations with TOPMed whole genome sequencing data and an epigenome-wide methylation array.<h4>Results</h4>Our results confirmed associations between FVIII and risk of incident CHD events and total mortality in AAs; mortality associations were largely independent of traditional risk factors. We also demonstrate an association of FVIII with incident heart failure, independent of B-type natriuretic peptide. Two genomic regions were strongly associated with FVIII (ABO and VWF). The index variant at VWF is specific to individuals of African descent and is distinct from the previously reported European VWF association signal. Epigenome-wide association analysis showed significant FVIII associations with several CpG sites in the ABO region. However, after adjusting for ABO genetic variants, ABO CpG sites were not significant.<h4>Conclusions</h4>Larger sample sizes of AAs will be required to discover additional genetic and epigenetic contributors to FVIII phenotypic variation, which may have consequences for CVD health disparities.

<h4>Aims</h4>We investigated the potential cooperative effects of carotenoid-producing Bacillus aquimaris SH6 and nonpigmented Bacillus subtilis SH23 on white-leg shrimp growth and health.<h4>Methods and results</h4>SH6, SH23 and a combination of both spores (1 × 10⁶  CFU per g pellet) were administered in shrimp. The growth rate (2·36% day^-1 ), red-colour score (25) and astaxanthin concentration (3·5 µg g^-1 shrimp) were maximum in two-spore-administered shrimp. Immune-related Rho mRNA expression level and phenoloxidase and superoxidase dismutase activities were higher in two-spore-administered shrimp than in control shrimp, with Rho mRNA expression level being 55-fold higher in two-spore-administered shrimp than in SH6-administered shrimp and phenoloxidase activity being 1·2-fold higher in two-spore-administered shrimp than in SH23-administered shrimp. Although live SH6 count was 2·7-fold lower, SH6 germination level was 3·5-fold higher in the combination group than in SH6 group.<h4>Conclusions</h4>When both SH6 and SH23 spores were administered, SH6 spore germination was enhanced and cooperative improvement was seen in growth, astaxanthin level and red-colour score of white-leg shrimp; however, immune-related parameters were induced in a noncooperative manner.<h4>Significance and impact of the study</h4>This is the first report showing the cooperative probiotic activities of Bacillus strains and their possible mechanisms in a shrimp model.

The physiological AKT-MTORC1 and AMPK signaling pathways are considered key nodes in the regulation of anabolism-catabolism, and particularly of macroautophagy/autophagy. Indeed, it is reported that these are altered processes in neurodegenerative proteinopathies such as Alzheimer disease (AD), mainly characterized by deposits of β-amyloid (Aβ) and hyperphosphorylated MAPT. These accumulations disrupt the optimal neuronal proteostasis, and hence, the recovery/enhancement of autophagy has been proposed as a therapeutic approach against these proteinopathies. The purpose of the present study was to characterize the modulation of autophagy by MTORC1 and AMPK signaling pathways in the highly specialized neurons, as well as their repercussions on Aβ production. Using a double transgenic mice model of AD, we demonstrated that MTORC1 inhibition, either <i>in vivo</i> or <i>ex vivo</i> (primary neuronal cultures), was able to reduce amyloid secretion through moderate autophagy induction in neurons. The pharmacological prevention of autophagy in neurons augmented the Aβ secretion and reversed the effect of rapamycin, confirming the anti-amyloidogenic effects of autophagy in neurons. Inhibition of AMPK with compound C generated the expected decrease in autophagy induction, though surprisingly did not increase the Aβ secretion. In contrast, increased activity of AMPK with metformin, AICAR, 2DG, or by gene overexpression did not enhance autophagy but had different effects on Aβ secretion: whereas metformin and 2DG diminished the secreted Aβ levels, AICAR and PRKAA1/AMPK gene overexpression increased them. We conclude that AMPK has a significantly different role in primary neurons than in other reported cells, lacking a direct effect on autophagy-dependent amyloidosis.<b>Abbreviations:</b> 2DG: 2-deoxy-D-glucose; Aβ: β-amyloid; ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AD: Alzheimer disease; AICAR: 5-aminoimidazole-4-carboxamide-1-β-riboside; AKT: AKT kinases group (AKT1 [AKT serine/threonine kinase 1], AKT2 and AKT3); AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; APP: amyloid beta precursor protein; APP/PSEN1: B6.Cg-Tg (APP^Swe, PSEN1dE9) 85Dbo/J; ATG: autophagy related; ATP: adenosine triphosphate; BafA1: bafilomycin A₁; CA: constitutively active; CGN: cerebellar granule neuron; CoC/compound C: dorsommorphin dihydrochloride; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gmax: GlutaMAX™; IN1: PIK3C3/VPS34-IN1; KI: kinase-inactive; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3; MAPT/TAU: microtubule associated protein tau; Metf: metformin; MRT: MRT68921; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RPS6KB1/S6K: ribosomal protein S6 (RPS6) kinase polypeptide 1; SCR: scramble; SQSTM1/p62: sequestosome 1; ULK1/2: unc-51 like autophagy activating kinase 1/2; WT: wild type.

<h4>Objective</h4>There are a limited number of studies evaluating iron overload in childhood leukemia by magnetic resonance imaging (MRI). The aim of this study was to determine liver iron content (LIC) by MRI in children with acute lymphoblastic leukemia (ALL) who had completed treatment and to compare those values with serum iron parameters.<h4>Materials and methods</h4>A total of 30 patients between the ages of 7 and 18 who had completed ALL treatment were included in the study. Serum iron parameters (serum iron, serum ferritin [SF], and total iron-binding capacity) and liver function tests were studied. R2 MRI was performed for determining LIC.<h4>Results</h4>Normal LIC was detected in 22 (63.4%) of the cases. Seven (23.3%) had mild and 1 (3.3%) had moderate liver iron deposition. In contrast, severe iron overload was not detected in any of the cases. LIC levels were correlated with the numbers of packed red blood cell (pRBC) transfusions (r=0.637, p<0.001), pRBC transfusion volume (r=0.449, p<0.013), SF levels (r=0.561, p=0.001), and transferrin saturation (r=0.353, p=0.044). In addition, a positive correlation was found between the number of pRBC transfusions and SF levels (r=0.595, p<0.001).<h4>Conclusion</h4>We showed that the frequency of liver iron deposition was low and clinically less significant after the end of treatment in childhood ALL patients. LIC was demonstrated to be related to SF and transfusion history. These findings support that SF and transfusion history may be used as references for monitoring iron accumulation or identifying cases for further examinations such as MRI.

With the gradual usage of carbon dots (CDs) in the area of antiviral research, attempts have been stepped up to develop new antiviral CDs with high biocompatibility and antiviral effects. In this study, a kind of highly biocompatible CDs (Gly-CDs) is synthesized from active ingredient (glycyrrhizic acid) of Chinese herbal medicine by a hydrothermal method. Using the porcine reproductive and respiratory syndrome virus (PRRSV) as a model, it is found that the Gly-CDs inhibit PRRSV proliferation by up to 5 orders of viral titers. Detailed investigations reveal that Gly-CDs can inhibit PRRSV invasion and replication, stimulate antiviral innate immune responses, and inhibit the accumulation of intracellular reactive oxygen species (ROS) caused by PRRSV infection. Proteomics analysis demonstrates that Gly-CDs can stimulate cells to regulate the expression of some host restriction factors, including DDX53 and NOS3, which are directly related to PRRSV proliferation. Moreover, it is found that Gly-CDs also remarkably suppress the propagation of other viruses, such as pseudorabies virus (PRV) and porcine epidemic diarrhea virus (PEDV), suggesting the broad antiviral activity of Gly-CDs. The integrated results demonstrate that Gly-CDs possess extraordinary antiviral activity with multisite inhibition mechanisms, providing a promising candidate for alternative therapy for PRRSV infection.

With the great success of anti-CTLA-4 and anti-PD-1 therapeutics in cancer immunotherapy, tumor necrosis factor receptor superfamily members have been recognized as ideal targets to provide co-stimulatory signals in combination with immune checkpoint blocking antibodies. Among these is OX40 (CD134), a co-stimulatory molecule expressed by activated immune cells. Recently, several anti-OX40 agonistic monoclonal antibodies, pogalizumab as the most advanced, have entered early phase clinical trials. Using a yeast platform and multiple screening methods, we identified a fully human anti-OX40 antibody (IBI101) with distinct modes of action. Unlike pogalizumab, IBI101 partially blocks the binding of OX40 to its ligand OX40L and exhibits both FcγR-dependent and independent agonistic activities in NF-κB luciferase reporter assays. IBI101 also promotes T cell activation and proliferation in vitro. These unique properties partially explain the more potent anti-tumor activity of IBI101 than that of pogalizumab in humanized NOG mice bearing LoVo tumors. In addition, IBI101 shows efficacious anti-tumor activity in mice when administrated alone or in combination with anti-PD-1 antibodies. In human OX40 knock-in mice bearing MC38 colon carcinoma, IBI101 treatment induces tumor antigen-specific CD8+ T-cell responses, decreases immunosuppressive regulatory T cells in tumor, and enhances the immune response to PD-1 inhibition. Preclinical studies of IBI101 in non-human primates demonstrate typical pharmacokinetic characteristics of an IgG antibody and no drug-related toxicity. Collectively, IBI101 has desirable preclinical attributes which support its clinical development for cancer treatment.

Chemotherapy-induced cardiovascular toxicity (CICT) is a well-established risk for cancer survivors and causes diseases such as heart failure, arrhythmia, vascular dysfunction, and atherosclerosis. As our knowledge of the precise cardiovascular risks of each chemotherapy agent has improved, it has become clear that genomics is one of the most influential predictors of which patients will experience cardiovascular toxicity. Most recently, GWAS-led, top-down approaches have identified novel genetic variants and their related genes that are statistically related to CICT. Importantly, the advent of human-induced pluripotent stem cell (hiPSC) models provides a system to experimentally test the effect of these genomic findings in vitro, query the underlying mechanisms, and develop novel strategies to mitigate the cardiovascular toxicity liabilities due to these mechanisms. Here we review the cardiovascular toxicities of chemotherapy drugs, discuss how these can be modeled in vitro, and suggest how these models can be used to validate genetic variants that predispose patients to these effects.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q₁₀, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Chronic pain is a major clinical problem of which the mechanisms are incompletely understood. Here, we describe the concept that PI16, a protein of unknown function mainly produced by fibroblasts, controls neuropathic pain. The spared nerve injury (SNI) model of neuropathic pain increases PI16 protein levels in fibroblasts in dorsal root ganglia (DRG) meninges and in the epi/perineurium of the sciatic nerve. We did not detect PI16 expression in neurons or glia in spinal cord, DRG, and nerve. Mice deficient in PI16 are protected against neuropathic pain. In vitro, PI16 promotes transendothelial leukocyte migration. In vivo, <i>Pi16</i>^-/- mice show reduced endothelial barrier permeability, lower leukocyte infiltration and reduced activation of the endothelial barrier regulator MLCK, and reduced phosphorylation of its substrate MLC2 in response to SNI. In summary, our findings support a model in which PI16 promotes neuropathic pain by mediating a cross-talk between fibroblasts and the endothelial barrier leading to barrier opening, cellular influx, and increased pain. Its key role in neuropathic pain and its limited cellular and tissue distribution makes PI16 an attractive target for pain management.

Therapeutic phlebotomy is the cornerstone of treatment for HFE haemochromatosis (HH). Current Australian Red Cross LifeBlood Service guidelines mandate measuring haemoglobin (Hb) levels prior to phlebotomy and if below 130 g/l in men or 120 g/l in women, donors are deferred from donating whole blood. Therapeutic donation below these levels may take place where both the treating doctor and a blood service medical officer approve. The aim of the current study was to determine whether adverse events are more frequent in those who undergo therapeutic phlebotomy below current Hb thresholds applied to volunteer therapeutic donors. A retrospective review of all therapeutic donations was undertaken for the financial year 2016-2017. The data were obtained through the Australian Red Cross Blood Service. Inclusion criteria were any donor between 16 and 70 years of age, weighing more than 50 kg and meeting blood service guidelines for donation. All adverse events recorded in an electronic quality system were obtained and associated with donor haemoglobin level. Statistical analyses were performed using analysis of variance or Fisher's exact test (GraphPad Prism). About 34 886 therapeutic phlebotomy donations occurred during 2016-2017, of whom the majority were referred for HH (34 089). In total, 365 of 34 886 donations (0·0105%) were complicated by an adverse event. A total of 305 (0·0087%) therapeutic donations occurred while below the lower limit of blood service Hb threshold for their respective genders. Of the donations that occurred below the blood service threshold, 3 of 305 (0·0098%) had an adverse event compared with 362 of 34 581 donations above the lower limit threshold (0·0105%, P = 0·99). The incidence of adverse events was not increased in the group which underwent therapeutic phlebotomy below the current Australian Red Cross Blood Service Hb threshold compared with those above threshold, indicating safety of treatment at Hb levels lower than currently recommended.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

Intestinal stem cells (ISCs) are confined to crypt bottoms and their progeny differentiate near crypt-villus junctions. Wnt and bone morphogenic protein (BMP) gradients drive this polarity, and colorectal cancer fundamentally reflects disruption of this homeostatic signaling. However, sub-epithelial sources of crucial agonists and antagonists that organize this BMP gradient remain obscure. Here, we couple whole-mount high-resolution microscopy with ensemble and single-cell RNA sequencing (RNA-seq) to identify three distinct PDGFRA⁺ mesenchymal cell types. PDGFRA(hi) telocytes are especially abundant at the villus base and provide a BMP reservoir, and we identified a CD81⁺ PDGFRA(lo) population present just below crypts that secretes the BMP antagonist Gremlin1. These cells, referred to as trophocytes, are sufficient to expand ISCs in vitro without additional trophic support and contribute to ISC maintenance in vivo. This study reveals intestinal mesenchymal structure at fine anatomic, molecular, and functional detail and the cellular basis for a signaling gradient necessary for tissue self-renewal.

Hepcidin peptide is crucial in the regulation of systemic iron availability controlling its uptake from the diet and its release from the body storage tissues. Hepcidin dysregulation causes different human disorders ranging from iron overload (e.g. hemochromatosis) to iron deficiency (e.g. anemia). Hepcidin excess is common in the Anemia of Chronic Diseases or Anemia of Inflammation and in the genetic form of anemia named IRIDA; the pharmacological downregulation of hepcidin in these disorders could improve the anemia. Commercial heparins were shown to be strong inhibitors of hepcidin expression, by interfering with BMP6/SMAD pathway. The non-anti-coagulant heparins, modified to abolish the anti-thrombin binding site, were equally potent and could be used to improve iron status. To perform its anti-hepcidin activity heparin needs 2O- and 6O-sulfation and an average molecular weight (MW) up to 4000-8000 Dalton, depending on the sulfation level. The pentosane polysulfate (PPS), which shares with heparin a high degree of sulfation, is a compound with low anti-coagulant activity that is already in use for pharmaceutical treatment. In the present work we analyzed the anti-hepcidin activity of PPS in vitro and in vivo. We found that it acts as a strong inhibitor of hepcidin expression in HepG2 cells with an effect already visible after 2-3 h of treatment. It also suppressed hepcidin in mice in a dose dependent manner after 3 h and with a significant redistribution of systemic iron without evident side effects. PPS is also able to abolish the LPS dependent hepcidin upregulation similarly to that showed for heparin derivatives. These results suggest PPS as an interesting compound to control hepcidin in vivo.

Multiple mechanisms have been suggested to confer to the pathophysiology of metabolic syndrome (MetS), however despite great interest from the scientific community, the exact contribution of each of MetS risk factors still remains unclear. The present study aimed to investigate molecular signatures in peripheral blood of individuals affected by MetS and different degrees of obesity. Metabolic health of 1204 individuals from 1000PLUS cohort was assessed, and 32 subjects were recruited to four study groups: MetS lean, MetS obese, "healthy obese", and healthy lean. Whole-blood transcriptome next generation sequencing with functional data analysis were carried out. MetS obese and MetS lean study participants showed the upregulation of genes involved in inflammation and coagulation processes: granulocyte adhesion and diapedesis (<i>p</i> < 0.0001, <i>p</i> = 0.0063), prothrombin activation pathway (<i>p</i> = 0.0032, <i>p</i> = 0.0091), coagulation system (<i>p</i> = 0.0010, <i>p</i> = 0.0155). The results for "healthy obese" indicate enrichment in molecules associated with protein synthesis (<i>p</i> < 0.0001), mitochondrial dysfunction (<i>p</i> < 0.0001), and oxidative phosphorylation (<i>p</i> < 0.0001). Our results suggest that MetS is related to the state of inflammation and vascular system changes independent of excess body weight. Furthermore, "healthy obese", despite not fulfilling the criteria for MetS diagnosis, seems to display an intermediate state with a lower degree of metabolic abnormalities, before they proceed to a full blown MetS.

Central nervous system (CNS) injury results in chronic scar formation that interferes with function and inhibits repair. Extracellular matrix (ECM) is prominent in the scar and potently regulates cell behavior. However, comprehensive information about the ECM proteome is largely lacking, and region- as well as injury-specific differences are often not taken into account. These aspects are the focus of our perspective on injury and scar formation. To highlight the importance of such comprehensive proteome analysis we include data obtained with novel analysis tools of the ECM composition in the scar and show the contribution of monocytes to the ECM composition after traumatic brain injury (TBI). Monocyte invasion was reduced using the CCR2-/- mouse line and step-wise de-cellularization and proteomics allowed determining monocyte-dependent ECM composition and architecture of the glial scar. We find significant reduction in the ECM proteins Tgm1, Itih (1,2, and 3), and Ftl in the absence of monocyte invasion. We also describe the scar ECM comprising zones with distinctive composition and show a subacute signature upon comparison to proteome obtained at earlier times after TBI. These results are discussed in light of injury-, region- and time-specific regulation of scar formation highlighting the urgent need to differentiate injury conditions and CNS-regions using comprehensive ECM analysis.

<b>Objective:</b> Long non-coding RNAs (lncRNAs) are critical to colorectal cancer (CRC) progression. In the current study, the objective was the exploration of the role played by lncRNA MIR4435-2HG in CRC proliferation and metastasis. <b>Methods:</b> lncRNA MIR4435-2HG expression and its association with CRC were analyzed using database and clinical specimens. The influences exerted by MIR4435-2HG on cell proliferating process, invading process, and migrating process of CRC were identified after MIR4435-2HG knockdown. The influences exerted by MIR4435-2HG on tumor growth and metastasis were assessed <i>in vivo</i>. The underlying mechanistic associations between MIR4435-2HG, microRNA miR-206, and the transcription factor Yes-associated protein 1 (YAP1) were assessed using bioinformatics and a luciferase reporter gene assay. <b>Results:</b> MIR4435-2HG was highly expressed in CRC tissue in contrast with that in regular tissues and displayed relations to poor prognosis. MIR4435-2HG knockdown could suppress CRC cell proliferation, invasion, and migration. Moreover, MIR4435-2HG knockdown inhibited CRC growth and liver metastasis <i>in vitro</i>. We found MIR4435-2HG knockdown reduced YAP1, CTGF, AREG, vimentin, Snail, Slug, and Twist expression but enhanced E-cadherin expression. Functionally, MIR4435-2HG acted as a competing endogenous RNA (ceRNA) to upregulate YAP1 by sponging miR-206. <b>Conclusions:</b> MIR4435-2HG promoted CRC growth and metastasis through miR-206/YAP1 axis and is likely to play prognostic marker roles and be therapeutically targeted in CRC.

<h4>Background</h4>The molecular mechanisms underlying chronic kidney disease (CKD) transition to end-stage renal disease (ESRD) and CKD acceleration of cardiovascular and other tissue inflammations remain poorly determined.<h4>Methods</h4>We conducted a comprehensive data analyses on 7 microarray datasets in peripheral blood mononuclear cells (PBMCs) from patients with CKD and ESRD from NCBI-GEO databases, where we examined the expressions of 2641 secretome genes (SG).<h4>Results</h4>1) 86.7% middle class (molecular weight >500 Daltons) uremic toxins (UTs) were encoded by SGs; 2) Upregulation of SGs in PBMCs in patients with ESRD (121 SGs) were significantly higher than that of CKD (44 SGs); 3) Transcriptomic analyses of PBMC secretome had advantages to identify more comprehensive secretome than conventional secretomic analyses; 4) ESRD-induced SGs had strong proinflammatory pathways; 5) Proinflammatory cytokines-based UTs such as IL-1β and IL-18 promoted ESRD modulation of SGs; 6) ESRD-upregulated co-stimulation receptors CD48 and CD58 increased secretomic upregulation in the PBMCs, which were magnified enormously in tissues; 7) M1-, and M2-macrophage polarization signals contributed to ESRD- and CKD-upregulated SGs; 8) ESRD- and CKD-upregulated SGs contained senescence-promoting regulators by upregulating proinflammatory IGFBP7 and downregulating anti-inflammatory TGF-β1 and telomere stabilizer SERPINE1/PAI-1; 9) ROS pathways played bigger roles in mediating ESRD-upregulated SGs (11.6%) than that in CKD-upregulated SGs (6.8%), and half of ESRD-upregulated SGs were ROS-independent.<h4>Conclusions</h4>Our analysis suggests novel secretomic upregulation in PBMCs of patients with CKD and ESRD, act synergistically with uremic toxins, to promote inflammation and potential disease progression. Our findings have provided novel insights on PBMC secretome upregulation to promote disease progression and may lead to the identification of new therapeutic targets for novel regimens for CKD, ESRD and their accelerated cardiovascular disease, other inflammations and cancers. (Total words: 279).

Damage to the intervertebral discs (IVDs) occurs due to aging or excessive mechanical stress, causing a series of IVD-related degenerative diseases, such as spinal disc herniation and spondylosis. These IVD-related diseases are difficult to cure, partially because the regeneration ability of IVDs is not sufficient. As a novel strategy for treatment of IVD-related diseases, mesenchymal stem cell transplantation to the damaged discs has been reported in animal studies. To further develop and improve this approach, it is necessary to gain a better understanding of the molecular network regulating IVD development by critical transcription factors. Recent findings reveal that during IVD development, nucleus pulposus and annuls fibrosus differentiation is coordinated by a series of transcription factors, such as <i>Mkx</i>, <i>Pax1</i>, <i>9</i>, <i>Shh</i>, <i>Foxa1, 2</i>, <i>T-Brachyury</i>, and <i>Sox5, 6, 9</i>. The combination of mesenchymal stem cell transplantation with the regulation of these molecules may provide a novel strategy for treatment of degenerative disc diseases.

The scope of the acid-mediated 3-component synthesis of thiadiazines was investigated. A selective functionalization of the six-membered heterocyclic core structure was accomplished by sequential alkylations, saponifications, and coupling reactions. Several new analogs of a dihydropyrimidinone Hsp70 chaperone agonist, MAL1-271, showed promising activity in a cell based model of Huntington's disease.

The synthesis and properties of various norbornadiene/quadricyclane (NBD/QC) fullerene hybrids are reported. By cyclopropanation of C₆₀ with malonates carrying the NBD scaffold a small library of NBD-fullerene monoadducts and NBD-fullerene hexakisadducts was established. The substitution pattern of the NBD scaffold, as well as the electron affinity of the fullerene core within these hybrid systems, has a pronounced impact on the properties of the corresponding energy rich QC derivatives. Based on this, the first direct photoisomerization of NBD-fullerene hybrids to their QC derivatives was achieved. Furthermore, it was possible to use the redox-active fullerene core of a QC-fullerene monoadduct to enable the back reaction to form the corresponding NBD-fullerene monoadduct. Combining these two processes enables switching between NBD and QC simply by changing the irradiation wavelength between 310 and 400 nm. Therefore, turning this usually photo/thermal switch into a pure photoswitch. This not only simplifies the investigation of the underlying processes of the NBD-QC interconversion within the system, but also renders such hybrids interesting for applications as molecular switches.

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9^e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.

<h4>Background</h4>There is a need for noninvasive methods for the diagnosis and monitoring of portal hypertension (PH).<h4>Purpose</h4>To 1) assess the correlation of liver and spleen T₁ and T_1ρ measurements with portal pressures in patients with chronic liver disease, and 2) to compare the diagnostic performance of the relaxation parameters with radiological assessment of PH.<h4>Study type</h4>Prospective.<h4>Subjects</h4>Twenty-five patients (M/F 16/9, mean age 56 years, range 21-78 years) undergoing portal pressure (hepatic venous pressure gradient [HVPG]) measurements.<h4>Field strength/sequence</h4>1.5T abdominal MRI scan, including T_1ρ and T₁ mapping.<h4>Assessment</h4>Liver and spleen T_1ρ and T₁ , radiological PH score, and (normalized) spleen length were evaluated.<h4>Statistical tests</h4>Spearman correlation of all MRI parameters with HVPG was assessed. The diagnostic performance of the assessed parameters for prediction of PH (HVPG ≥5 mmHg) and clinically significant PH (CSPH, HVPG ≥10 mmHg) was determined by receiver operating characteristic (ROC) analysis.<h4>Results</h4>The mean HVPG measurement was 7.8 ± 5.3 mmHg (PH, n = 18 [72%] including CSPH, n = 9 [36%]). PH score, (normalized) spleen length and spleen T_1ρ significantly correlated with HVPG, with the strongest correlation found for spleen T_1ρ (r = 0.613, P = 0.001). Spleen T_1ρ was the only parameter that showed significant diagnostic performance for assessment of PH (area under the curve [AUC] 0.817, P = 0.015) and CSPH (AUC = 0.778, P = 0.024). Normalized spleen length also showed significant diagnostic performance for prediction of CSPH, with a slightly lower AUC (= 0.764, P = 0.031). The radiological PH score, T_1ρ and T₁ of the liver and T₁ of the spleen, did not show significant diagnostic performance for assessment of CSPH (P > 0.075).<h4>Data conclusion</h4>Spleen T_1ρ showed a significant correlation with portal pressure and showed improved diagnostic performance for prediction of CSPH compared to radiological assessment. These initial results need confirmation in a larger cohort.<h4>Level of evidence</h4>1 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;52:787-794.

<h4>Background</h4>Stratification of the severity of infection is currently based on the Sequential Organ Failure Assessment (SOFA) score, which is difficult to calculate outside the ICU. Biomarkers could help to stratify the severity of infection in surgical patients.<h4>Methods</h4>Levels of ten biomarkers indicating endothelial dysfunction, 22 indicating emergency granulopoiesis, and six denoting neutrophil degranulation were compared in three groups of patients in the first 12 h after diagnosis at three Spanish hospitals.<h4>Results</h4>There were 100 patients with infection, 95 with sepsis and 57 with septic shock. Seven biomarkers indicating endothelial dysfunction (mid-regional proadrenomedullin (MR-ProADM), syndecan 1, thrombomodulin, angiopoietin 2, endothelial cell-specific molecule 1, vascular cell adhesion molecule 1 and E-selectin) had stronger associations with sepsis than infection alone. MR-ProADM had the highest odds ratio (OR) in multivariable analysis (OR 11·53, 95 per cent c.i. 4·15 to 32·08; P = 0·006) and the best area under the curve (AUC) for detecting sepsis (0·86, 95 per cent c.i. 0·80 to 0·91; P < 0·001). In a comparison of sepsis with septic shock, two biomarkers of neutrophil degranulation, proteinase 3 (OR 8·09, 1·34 to 48·91; P = 0·028) and lipocalin 2 (OR 6·62, 2·47 to 17·77; P = 0·002), had the strongest association with septic shock, but lipocalin 2 exhibited the highest AUC (0·81, 0·73 to 0·90; P < 0·001).<h4>Conclusion</h4>MR-ProADM and lipocalin 2 could be alternatives to the SOFA score in the detection of sepsis and septic shock respectively in surgical patients with infection.

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.

Chronic inflammation during pregnancy (e.g., preeclampsia, diabetes) is linked to increased risk for offspring neurodevelopmental disorders such as autism spectrum disorder (ASD). However, mediators of such exposures that could be targeted with maternal intervention are unclear, as few chronic gestational inflammation models have been tested. One potential mediator is interleukin-17 (IL-17), a pro-inflammatory cytokine implicated in neurodevelopmental disorders and gestational disease. To test chronic maternal IL-17 impacts on offspring, C57BL/6J dams were administered IL-17A continuously throughout pregnancy. Offspring were assessed for body weight; cortical volume, gene expression, and cellular composition; and adult behavior. IL-17A-condition offspring exhibited decreased somatic and cortical size at embryonic day 18 (E18) and as adults. mRNA sequencing of E18 cortex revealed 320 differentially expressed genes in males, but none in females. These were significantly enriched for ASD (Simons Foundation Autism Research Initiative), synaptic, and cell cycle genes. By adulthood, neocortical glial cell density and gene expression were decreased, while GABAergic synaptic gene expression was increased in males. Furthermore, IL-17A-condition male but not female offspring exhibited reduced anxiety-like behavior. Social approach deficits in males were negatively correlated with neocortical GABAergic synaptic gene expression. Chronic gestational IL-17A was sufficient to cause ASD-like phenotypes early and persistently in male offspring. This echoes the male bias, altered cortical development, and behavioral findings in ASD, suggesting that chronic maternal IL-17 contributes to offspring ASD pathogenesis. Furthermore, the trajectory from embryonically dysregulated synaptic and cell cycle genes to disrupted adult glia, inhibitory synapses, and behavior suggests a mechanism for chronic maternal IL-17 effects on offspring.

GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders^1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric G_s protein², but it is unclear how GPR52 and G_s couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a G_s-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR52³. A fully active state is achieved when G_s is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.

Malaria is an old scourge of humankind and has a large negative impact on the economic development of affected communities. Recent success in malaria control and reduction of mortality seems to have stalled emphasizing that our current intervention tools need to be complemented by malaria vaccines. Different populations of unconventional T cells such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cells are gaining attention in the field of malaria immunology. Significant advances in our basic understanding of unconventional T cell biology in rodent malaria models have been made, however, their roles in humans during malaria are less clear. Unconventional T cells are abundant in skin, gut and liver tissues, and long-lasting expansions and functional alterations were observed upon malaria infection in malaria naïve and malaria pre-exposed volunteers. Here, we review the current understanding of involvement of unconventional T cells in anti-Plasmodium falciparum immunity and highlight potential future research avenues.

Pulmonary hypertension (PH) has been described in myelofibrosis (MF), but it is rare and typically found in advanced disease. Although the etiology of PH in MF is unclear, early predictors may be detected by echocardiogram. The goals of our study were to evaluate the prevalence of PH as determined by echocardiography in a cohort of MF patients and to identify clinical risk factors for PH. We performed a retrospective review of MF patients from October 2015 to May 2017 at MD Anderson Cancer Center in the ambulatory clinic, and those with echocardiogram were included. Clinical, echocardiographic, and laboratory data were reviewed. Patients with and without PH were compared using a chi-square or Fisher's exact test, and logistic regression was performed with an outcome variable of PH. There were 143 patients with MF who underwent echocardiogram, and 20 (14%) had echocardiographic findings consistent with PH. Older age, male gender, hypertension, hyperlipidemia, coronary artery disease, dyspnea, hematocrit, brain natriuretic peptide (BNP), and N-terminal prohormone BNP (NT-proBNP) were significantly different between those without PH and those with PH (p < 0.05). Female gender was protective (OR 0.21, 95% CI 0.049-0.90, p = 0.035), and NT-proBNP was a significant clinical predictor of PH (OR 1.07, CI 1.02 = 1.12, p = 0.006). PH in MF is lower than previously reported in our MF cohort, but many patients had cardiac comorbidities. PH due to left-sided heart disease may be underestimated in MF. Evaluation of respiratory symptoms and elevated NT-proBNP should prompt a baseline echocardiogram. Early detection of PH with a multidisciplinary approach may allow treatment of reversible etiologies.

<h4>Background</h4>Mesenchymal stem/stromal cells (MSCs) from the decidua basalis (DBMSCs) of the human placenta have important functions that make them potential candidates for cellular therapy. Previously, we showed that DBMSC functions do not change significantly in a high oxidative stress environment, which was induced by hydrogen peroxide (H₂O₂) and immune cells. Here, we studied the consequences of glucose, another oxidative stress inducer, on the phenotypic and functional changes in DBMSCs.<h4>Methods</h4>DBMSCs were exposed to a high level of glucose, and its effect on DBMSC phenotypic and functional properties was determined. DBMSC expression of oxidative stress and immune molecules after exposure to glucose were also identified.<h4>Results</h4>Conditioning of DBMSCs with glucose improved their adhesion and invasion. Glucose also increased DBMSC expression of genes with survival, proliferation, migration, invasion, anti-inflammatory, anti-chemoattractant and antimicrobial properties. In addition, DBMSC expression of B7H4, an inhibitor of T cell proliferation was also enhanced by glucose. Interestingly, glucose modulated DBMSC expression of genes involved in insulin secretion and prevention of diabetes.<h4>Conclusion</h4>These data show the potentially beneficial effects of glucose on DBMSC functions. Preconditioning of DBMSCs with glucose may therefore be a rational strategy for increasing their therapeutic potential by enhancing their engraftment efficiency. In addition, glucose may program DBMSCs into insulin producing cells with ability to counteract inflammation and infection associated with diabetes. However, future in vitro and in vivo studies are essential to investigate the findings of this study further.

Neurodegenerative diseases (NDDs) comprise conditions with impaired neuronal function and loss and may be associated with a build-up of aggregated proteins with altered physicochemical properties (misfolded proteins). There are many disorders, and causes include gene mutations, infections, or exposure to toxins. The autophagy pathway is involved in the removal of unwanted proteins and organelles through lysosomes. While lysosomal storage disorders have been described for many years, it is now recognised that perturbations of the autophagy pathway itself can also lead to neurodegenerative disease. These include monogenic disorders of key proteins involved in the autophagy pathway, and disorders within pathways that critically control autophagy through monitoring of the supply of nutrients (mTORC1 pathway) or of energy supply in cells (AMPK pathway). This review focuses on childhood-onset neurodegenerative disorders with perturbed autophagy, due to defects in the autophagy pathway, or in upstream signalling via mTORC1 and AMPK. The review first provides a short description of autophagy, as related to neurons. It then examines the extended role of autophagy in neuronal function, plasticity, and memory. There follows a description of each step of the autophagy pathway in greater detail, illustrated with examples of diseases grouped by the stage of their perturbation of the pathway. Each disease is accompanied by a short clinical description, to illustrate the diversity but also the overlap of symptoms caused by perturbation of key proteins necessary for the proper functioning of autophagy. Finally, there is a consideration of current challenges that need addressing for future therapeutic advances.

We conducted a two-sample Mendelian randomization study to explore the associations of iron status with overall cancer and 22 site-specific cancers. Single-nucleotide polymorphisms for iron status were obtained from a genome-wide association study of 48,972 European-descent individuals. Summary-level data for breast and other cancers were obtained from the Breast Cancer Association Consortium and UK Biobank. Genetically predicted iron status was positively associated with liver cancer and inversely associated with brain cancer but not associated with overall cancer or the other 20 studied cancer sites at <i>p</i> < 0.05. The odds ratios of liver cancer were 2.45 (95% CI, 0.81, 7.45; <i>p</i> = 0.11), 2.11 (1.16, 3.83; <i>p</i> = 0.02), 10.89 (2.44, 48.59; <i>p</i> = 0.002) and 0.30 (0.17, 0.53; <i>p</i> = 2 × 10^-5) for one standard deviation increment of serum iron, transferrin saturation, ferritin and transferrin levels, respectively. For brain cancer, the corresponding odds ratios were 0.69 (0.48, 1.00; <i>p</i> = 0.05), 0.75 (0.59, 0.97; <i>p</i> = 0.03), 0.41 (0.20, 0.88; <i>p</i> = 0.02) and 1.49 (1.04, 2.14; <i>p</i> = 0.03). Genetically high iron status was positively associated with liver cancer and inversely associated with brain cancer.

Platelet-rich plasma (PRP) has attracted much attention for the treatment of articular cartilage defects or wounds due to its intrinsic content of growth factors relevant for tissue repair. However, the short residence time of PRP in vivo, due to the action of lytic enzymes, its weak mechanical properties and the consequent short-term release of bioactive factors has restricted its application and efficacy. The present work aimed at designing new formulation strategies for PRP, based on the use of platelet concentrate (PC)-loaded hydrogels or interpenetrating polymer networks, directed at improving mechanical stability and sustaining the release of bioactive growth factors over a prolonged time-span. The interpenetrating hydrogels comprised two polymer networks interlaced on a molecular scale: (<i>a</i>) a first covalent network of thermosensitive and biodegradable vinyl sulfone bearing p(hydroxypropyl methacrylamide-lacate)-polyethylene glycol triblock copolymers, tandem cross-linked by thermal gelation and Michael addition when combined with thiolated hyaluronic acid, and (<i>b</i>) a second network composed of cross-linked fibrin. The PC-loaded hydrogels, instead, was formed only by network (<i>a</i>). All the designed and successfully synthesized formulations greatly increased the stability of PRP in vitro, leading to significant increase in degradation time and storage modulus of PRP gel. The resulting viscoelastic networks showed the ability to controllably release platelet derived growth factor and transforming growth factr β1, and to improve the tissue adhesiveness of PRP. The newly developed hydrogels show great potential for application in the field of wound healing, cartilage repair and beyond.

Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, αB-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization.

Oxidative stress is a common culprit of several conditions associated with male fertility. High levels of reactive oxygen species (ROS) promote impairment of sperm quality mainly by decreasing motility and increasing the levels of DNA oxidation. Oxidative stress is a common feature of environmental pollutants, chemotherapy and other chemicals, smoke, toxins, radiation, and diseases that can have negative effects on fertility. Peroxiredoxins (PRDXs) are antioxidant enzymes associated with the protection of mammalian spermatozoa against oxidative stress and the regulation of sperm viability and capacitation. In the present study, we aimed to determine the long-term effects of oxidative stress in the testis, epididymis and spermatozoa using the rat model. Adult male rats were treated with tert-butyl hydroperoxide (t-BHP) or saline (control group), and reproductive organs and spermatozoa were collected at 3, 6, and 9 weeks after the end of treatment. We determined sperm DNA oxidation and motility, and levels of lipid peroxidation and protein expression of antioxidant enzymes in epididymis and testis. We observed that cauda epididymal spermatozoa displayed low motility and high DNA oxidation levels at all times. Lipid peroxidation was higher in caput and cauda epididymis of treated rats at 3 and 6 weeks but was similar to control levels at 9 weeks. PRDX6 was upregulated in the epididymis due to t-BHP; PRDX1 and catalase, although not significant, followed similar trend of increase. Testis of treated rats did not show signs of oxidative stress nor upregulation of antioxidant enzymes. We concluded that t-BHP-dependent oxidative stress promoted long-term changes in the epididymis and maturing spermatozoa that result in the impairment of sperm quality.

Fine-tuning loading and release of therapeutic and imaging agents associated with polymeric matrices is a fundamental step in the preclinical development of novel nanomedicines. Here, 1,000 × 400 nm Discoidal Polymeric Nanoconstructs (DPNs) were realized via a top-down, template-based fabrication approach, mixing together poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol)-diacrylate (PEG-DA) chains in a single polymer paste. Two different loading strategies were tested, namely the "direct loading" and the "absorption loading." In the first case, the agent was directly mixed with the polymeric paste to realize DPNs whereas, in the second case, DPNs were first lyophilized and then rehydrated upon exposure to a concentrated aqueous solution of the agent. Under these two loading conditions, the encapsulation efficiencies and release profiles of different agents were systematically assessed. Specifically, six agents were realized by conjugating lipid chains (DSPE) or polymeric chains (PEG) to the near-infrared imaging molecule Cy5 (DSPE-Cy5 A and DSPE-Cy5 B); the chemotherapeutic molecules methotrexate (DSPE-MTX and PEG-MTX) and doxorubicin (LA-DOX and DSPE-DOX). Moderately hydrophobic compounds with low molecular weights (MW) returned encapsulation efficiencies as high as 80% for the absorption loading. In general, direct loading was associated with encapsulation efficiencies lower than 1%. The agent hydrophobicity and MW were shown to be critical also in tailoring the release profiles from DPNs. On triple-negative breast cancer cells (MDA-MB-231), absorption loaded DOX-DPNs showed cytotoxic activities comparable to free DOX but slightly delayed in time. Preliminary <i>in vivo</i> studies demonstrated the high stability of Cy5-DPNs. Collectively, these results demonstrate that the pharmacological properties of DPNs can be finely optimized by changing the loading strategies (direct vs. absorption) and compound attributes (hydrophobicity and molecular weight).

<h4>Background</h4>Lung cancer is the most common cancer and the most common cause of cancer-related death worldwide. However, the molecular mechanism of its development is unclear. It is imperative to identify more novel biomarkers.<h4>Methods</h4>Two datasets (GSE70880 and GSE113852) were downloaded from the Gene Expression Omnibus (GEO) database and used to identify the differentially expressed genes (DEGs) between lung cancer tissues and normal tissues. Then, we constructed a competing endogenous RNA (ceRNA) network and a protein-protein interaction (PPI) network and performed gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and survival analyses to identify potential biomarkers that are related to the diagnosis and prognosis of lung cancer.<h4>Results</h4>A total of 41 lncRNAs and 805 mRNAs were differentially expressed in lung cancer. The ceRNA network contained four lncRNAs (CLDN10-AS1, SFTA1P, SRGAP3-AS2, and ADAMTS9-AS2), 21 miRNAs, and 48 mRNAs. Functional analyses revealed that the genes in the ceRNA network were mainly enriched in cell migration, transmembrane receptor, and protein kinase activity. mRNAs DLGAP5, E2F7, MCM7, RACGAP1, and RRM2 had the highest connectivity in the PPI network. Immunohistochemistry (IHC) demonstrated that mRNAs DLGAP5, MCM7, RACGAP1, and RRM2 were upregulated in lung adenocarcinoma (LUAD). Survival analyses showed that lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 were associated with the prognosis of LUAD.<h4>Conclusion</h4>lncRNAs CLDN10-AS1, SFTA1P, and ADAMTS9-AS2 might be the biomarkers of LUAD. For the first time, we confirmed the important role of lncRNA CLDN10-AS1 in LUAD.

<h4>Introduction</h4>Respiratory distress is an uncommon clinical event after caesarean section and occurs due to pulmonary thromboembolism. Various causes of pulmonary thromboembolism are thrombophlebitis, ovarian venous thrombosis and mesenteric vein thrombosis.<h4>Presentation of case</h4>We report a case of 30 year old female who presented with respiratory distress after eight days of uneventful caesarian section. On emergency explorative laparotomy, small gut was found to be gangrenous, so resection of the segment was performed. On histopathological examination, there was ischaemic necrosis of bowel with presence of large thrombus in mesenteric vessel. On correlating radiological findings of pulmonary thromboembolism and mesenteric vessel thrombosis with bad obstetric history, a possibility of Antiphospholipid syndrome (APS) was suggested in this case. Unfortunately, patient died the day following laparotomy so there was insufficient time to evaluate the patient for thrombophilic disorders.<h4>Discussion</h4>Pregnancy and perpeurium are associated with higher risk of thrombosis as these are hypercoagulable states. Operative delivery and history of thrombophilia in previous pregnancies (APS) are other predisposing factors which lead to increased thrombotic state and pulmonary thromboembolism.<h4>Conclusion</h4>High index of suspicion for thrombophilic disorders is required in postpartum patients presenting with respiratory distress as prompt diagnosis and urgent intervention can save patient's life.

<h4>Background and aims</h4>Patients with cirrhosis have a rebalanced hemostasis, often with normal or elevated thrombin-generating (TG) capacity in plasma. Whole blood (WB) TG allows faster determination and, importantly, includes the influence of all circulating blood cells. We aimed to study the TG profile of patients with cirrhosis in WB and in platelet poor plasma.<h4>Methods</h4>Thrombin-generating capacity in WB and plasma were assessed with a near-patient WB-TG assay and the calibrated automated thrombinography assay, respectively. TG assays were tested in presence and absence of thrombomodulin. Conventional coagulation tests were also performed.<h4>Results</h4>Thirty-four patients with cirrhosis and twenty-two controls were analyzed. Compared with controls, patients had substantially deranged results in conventional coagulation tests. Comparable WB-TG capacity (endogenous thrombin potential until peak, ETPp) but significantly lower peak thrombin were found in patients, and these results persisted when thrombomodulin was present. TG of the patients was more resistant to thrombomodulin than controls in both WB and plasma, although the inhibitory effect of thrombomodulin was drastically weaker in WB than in plasma. The peak of WB-TG in patients correlated moderately with their hematocrit and platelet count. Significant correlations were found between TG results in WB and plasma.<h4>Conclusions</h4>The WB-TG assay shows a normal to hypocoagulable state in patients with cirrhosis with a decreased anticoagulant activity of TM compared to plasma-TG. The clinical value of this assay needs further validation.

Fast excitatory synaptic transmission in the mammalian brain is largely mediated by AMPA-type ionotropic glutamate receptors (AMPARs), which are activated by the neurotransmitter glutamate. In synapses, the function of AMPARs is tuned by their auxiliary subunits, a diverse set of membrane proteins associated with the core pore-forming subunits of the AMPARs. Each auxiliary subunit provides distinct functional modulation of AMPARs, ranging from regulation of trafficking to shaping ion channel gating kinetics. Understanding the molecular mechanism of the function of these complexes is key to decoding synaptic modulation and their global roles in cognitive activities, such as learning and memory. Here, we review the structural and molecular complexity of AMPAR-auxiliary subunit complexes, as well as their functional diversity in different brain regions. We suggest that the recent structural information provides new insights into the molecular mechanisms underlying synaptic functions of AMPAR-auxiliary subunit complexes.

Pediatric sepsis is a leading cause of morbidity and mortality in children. One of the most common and devastating morbidities is sepsis-related acute kidney injury (AKI). AKI was traditionally thought to be related to low perfusion and acute tubular necrosis. However, little acute tubular necrosis can be found following septic AKI, and little is known about the mechanism of septic AKI. Olfactomedin-4 (OLFM4) is a secreted glycoprotein that marks a subset of neutrophils. Increased expression of OLFM4 in the blood is associated with worse outcomes in sepsis. Here, we investigated a pediatric model of murine sepsis using murine pups to investigate the mechanisms of OLFM4 in sepsis. When sepsis was induced in murine pups, survival was significantly increased in OLFM4-null pups. Immunohistochemistry at 24 h after the induction of sepsis demonstrated increased expression of OLFM4 in the kidney, which was localized to the loop of Henle. Renal cell apoptosis and plasma creatinine were significantly increased in wild-type versus OLFM4-null pups. Finally, bone marrow transplant suggested that increased OLFM4 in the kidney reflects local production rather than filtered from the plasma. These results demonstrate renal expression of OLFM4 for the first time and suggest that a kidney-specific mechanism may contribute to survival differences in OLFM4-null animals.

<h4>Background and aims</h4>Chronic liver disease (CLD) and cirrhosis are leading causes of death globally with the burden of disease rising significantly over the past several decades. Defining the etiology of liver disease is important for understanding liver disease epidemiology, healthcare planning, and outcomes. The aim of this study was to validate a hierarchical algorithm for CLD and cirrhosis etiology in administrative healthcare data.<h4>Methods</h4>Consecutive patients with CLD or cirrhosis attending an outpatient hepatology clinic in Ontario, Canada from 05/01/2013-08/31/2013 underwent detailed chart abstraction. Gold standard liver disease etiology was determined by an attending hepatologist as hepatitis C (HCV), hepatitis B (HBV), alcohol-related, non-alcoholic fatty liver disease (NAFLD)/cryptogenic, autoimmune or hemochromatosis. Individual data was linked to routinely collected administrative healthcare data at ICES. Diagnostic accuracy of a hierarchical algorithm incorporating both laboratory and administrative codes to define etiology was evaluated by calculating sensitivity, specificity, positive (PPV) and negative predictive values (NPV), and kappa's agreement.<h4>Results</h4>442 individuals underwent chart abstraction (median age 53 years, 53% cirrhosis, 45% HCV, 26% NAFLD, 10% alcohol-related). In patients with cirrhosis, the algorithm had adequate sensitivity/PPV (>75%) and excellent specificity/NPV (>90%) for all etiologies. In those without cirrhosis, the algorithm was excellent for all etiologies except for hemochromatosis and autoimmune diseases.<h4>Conclusions</h4>A hierarchical algorithm incorporating laboratory and administrative coding can accurately define cirrhosis etiology in routinely collected healthcare data. These results should facilitate health services research in this growing patient population.

Optogenetics has recently gained recognition as a biological technique to control the activity of cells using light stimulation. Many studies have applied optogenetics to cell lines in the central nervous system because it has the potential to elucidate neural circuits, treat neurological diseases and promote nerve regeneration. There have been fewer studies on the application of optogenetics in the peripheral nervous system. This review introduces the basic principles and approaches of optogenetics and summarizes the physiology and mechanism of opsins and how the technology enables bidirectional control of unique cell lines with superior spatial and temporal accuracy. Further, this review explores and discusses the therapeutic potential for the development of optogenetics and its capacity to revolutionize treatment for refractory epilepsy, depression, pain, and other nervous system disorders, with a focus on neural regeneration, especially in the peripheral nervous system. Additionally, this review synthesizes the latest preclinical research on optogenetic stimulation, including studies on non-human primates, summarizes the challenges, and highlights future perspectives. The potential of optogenetic stimulation to optimize therapy for peripheral nerve injuries (PNIs) is also highlighted. Optogenetic technology has already generated exciting, preliminary evidence, supporting its role in applications to several neurological diseases, including PNIs.

Huntington Disease (HD) is an inherited movement disorder caused by expanded CAG repeats in the Huntingtin gene. We have used single nucleus RNASeq (snRNASeq) to uncover cellular phenotypes that change in the disease, investigating single cell gene expression in cingulate cortex of patients with HD and comparing the gene expression to that of patients with no neurological disease. In this study, we focused on astrocytes, although we found significant gene expression differences in neurons, oligodendrocytes, and microglia as well. In particular, the gene expression profiles of astrocytes in HD showed multiple signatures, varying in phenotype from cells that had markedly upregulated metallothionein and heat shock genes, but had not completely lost the expression of genes associated with normal protoplasmic astrocytes, to astrocytes that had substantially upregulated glial fibrillary acidic protein (GFAP) and had lost expression of many normal protoplasmic astrocyte genes as well as metallothionein genes. When compared to astrocytes in control samples, astrocyte signatures in HD also showed downregulated expression of a number of genes, including several associated with protoplasmic astrocyte function and lipid synthesis. Thus, HD astrocytes appeared in variable transcriptional phenotypes, and could be divided into several different "states", defined by patterns of gene expression. Ultimately, this study begins to fill the knowledge gap of single cell gene expression in HD and provide a more detailed understanding of the variation in changes in gene expression during astrocyte "reactions" to the disease.

<h4>Purpose</h4>Prostate cancers with mutations in genes involved in homologous recombination (HR), most commonly BRCA2, respond favorably to PARP inhibition and platinum-based chemotherapy. We investigated whether other prostate tumors that do not harbor deleterious mutations in these particular genes can similarly be deficient in HR, likely rendering those sensitive to HR-directed therapies.<h4>Experimental design</h4>Homologous recombination deficiency (HRD) levels can be estimated using various mutational signatures derived from next-generation sequencing data. We used this approach on whole-genome sequencing (WGS; <i>n</i> = 311) and whole-exome sequencing (WES) data (<i>n</i> = 498) of both primary and metastatic prostate adenocarcinomas to determine whether prostate cancer cases display clear signs of HRD in somatic tumor biopsies.<h4>Results</h4>Known BRCA-deficient samples showed all previously described HRD-associated mutational signatures in the WGS data. HRD-associated mutational signatures were also detected in a subset of patients who did not harbor germline or somatic mutations in BRCA1/2 or other HR-related genes. Similar results, albeit with lower sensitivity and accuracy, were also obtained from WES data.<h4>Conclusions</h4>These findings may expand the number of cases likely to respond to PARP inhibitor treatment. On the basis of the HR-associated mutational signatures, 5% to 8% of localized prostate cancer cases may be good candidates for PARP-inhibitor treatment (including those with BRCA1/2 mutations).

Transfer RNA (tRNA) genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast <i>Saccharomyces cerevisiae</i> tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon <i>Salmonella</i> infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the <i>Salmonella</i>-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that <i>Salmonella</i> uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by <i>Salmonella</i> and other pathogens.<b>IMPORTANCE</b> Infection research routinely employs <i>in vitro</i> cell cultures or <i>in vivo</i> mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the <i>in vivo</i>-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen <i>Salmonella enterica</i> serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed <i>Salmonella</i>-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.

<h4>Background</h4>Maternal obesity prior to or during pregnancy influences fetal growth, predisposing the offspring to increased risk for obesity across the life course. Placental epigenetic mechanisms may underlie these associations. We conducted an epigenome-wide association study to identify placental DNA methylation changes associated with maternal prepregnancy body mass index (BMI) and rate of gestational weight gain at first (GWG1), second (GWG2), and third trimester (GWG3).<h4>Method</h4>Participants of the NICHD Fetal Growth Studies with genome-wide placental DNA methylation (n = 301) and gene expression (n = 75) data were included. Multivariable-adjusted regression models were used to test the associations of 1 kg/m² increase in prepregnancy BMI or 1 kg/week increase in GWG with DNA methylation levels. Genes harboring top differentially methylated CpGs (FDR P < 0.05) were evaluated for placental gene expression. We assessed whether DNA methylation sites known to be associated with BMI in child or adult tissues, were also associated with maternal prepregnancy BMI in placenta.<h4>Results</h4>Prepregnancy BMI was associated with DNA methylation at cg14568196[EGFL7], cg15339142[VETZ], and cg02301019[AC092377.1] (FDR P < 0.05, P ranging from 1.4 × 10^-10 to 1.7 × 10^-9). GWG1 or GWG2 was associated with DNA methylation at cg17918270[MYT1L], cg20735365[DLX5], and cg17451688[SLC35F3] (FDR P < 0.05, P ranging from 6.4 × 10^-10 to 1.2 × 10^-8). Both prepregnancy BMI and DNA methylation at cg1456819 [EGFL7] were negatively correlated with EGFL7 expression in placenta (P < 0.05). Several CpGs previously implicated in obesity traits in children and adults were associated with prepregnancy BMI in placenta. Functional annotations revealed that EGFL7 is highly expressed in placenta and the differentially methylated CpG sites near EGFL7 and VEZT were cis-meQTL targets in blood.<h4>Conclusions</h4>We identified placental DNA methylation changes at novel loci associated with prepregnancy BMI and GWG. The overlap between CpGs associated with obesity traits in placenta and other tissues in children and adults suggests that epigenetic mechanisms in placenta may give insights to early origins of obesity.

Iron is central to multiple biological pathways, and treatment of non-anaemic absolute iron deficiency (NAID) is beneficial in certain conditions. However, it is unknown if NAID is associated with increased mortality in older adults. A nationally representative sample of 4451 older adults from the English Longitudinal Study of Ageing was used. NAID was defined as serum ferritin < 30 μg/l and haemoglobin ≥ 120 g/l (women) or ≥ 130 g/l (men). Cumulative mortality was estimated by Kaplan-Meier method. Unadjusted and adjusted hazard ratios (HRs) of mortality were calculated using Cox proportional hazards regression models. Baseline NAID prevalence was 8·8% (95% confidence interval [CI] 8·0-9·7%); 10·9% (95% CI 9·7-12·3%) for women and 6·35% for men (95% CI 5·3-7·5%). The HR for mortality for individuals with NAID compared with non-anaemic individuals without iron deficiency over the 14-year follow-up was 1·58 (95% CI 1·29-1·93). This association was independent of all identified demographic, health-related and biological covariates, and robust in multiple sensitivity analyses. In older adults in England, NAID is common and associated with an increased mortality rate compared to non-anaemic individuals with normal serum ferritin. The association is principally driven by an excess mortality in women.

<h4>Introduction</h4>Hand osteoarthritis (HOA) is a highly prevalent disabling joint disease. The current management regimens are limited. Potentially as a consequence, many people turn to complementary and alternative medicines for symptomatic relief. A combination of two or more supplements is common in clinical practice; however, evidence for the efficacy of this approach is lacking. The aim of this study is to investigate the efficacy of a supplement combination for treating symptomatic HOA in comparison to placebo.<h4>Methods and analysis</h4>The RADIANT study is an internet-based, parallel, superiority, double-blind, placebo-controlled, randomised, two-arm clinical trial. A participatory design is used to facilitate the study procedures. One hundred and six participants aged over 40 years with painful HOA and structural change on X-ray (Kellgren and Lawrence grade (KLG) ≥2) will be recruited from the community and randomly allocated to receive either a supplement combination composed of: (1) combined supplement containing <i>Boswellia serrata</i> extract, pine bark extract and methylsulfonylmethane and (2) curcumin or placebo for 12 weeks. The primary outcome will be 12-week change in hand pain on a visual analogue scale (VAS). Main secondary outcomes include adverse events, change in hand function, patient global assessment of disease activity and quality of life. A range of additional measures will be recorded, and an individual patient placebo response will be performed. The primary analysis will be conducted using an intention-to-treat approach. Adverse events will be monitored weekly throughout the study.<h4>Ethics and dissemination</h4>This protocol has been approved by the University of Sydney Human Research Ethics Committee (HREC No. 2018/766). Dissemination will occur through conferences, social media, scientific publications and PhD thesis.<h4>Trial registration number</h4>Australian New Zealand Clinical Trials Registry (ACTRN12619000835145); Pre-results.

Radiation-induced normal tissue toxicity often limits the curative treatment of cancer. Moreover, normal tissue relative biological effectiveness data for high-linear energy transfer particles are urgently needed. We propose a strategy based on transcriptome analysis of patient-derived human intestinal organoids (HIO) to determine molecular surrogates for radioresponse of gastrointestinal (GI) organs at risk in a personalized manner. HIO were generated from induced pluripotent stem cells (iPSC), which were derived from skin biopsies of three patients, including two patients with FANCA deficiency as a paradigm for enhanced radiosensitivity. For the two Fanconi anemia (FA) patients (HIO-104 and 106, previously published as FA-A#1 IND-iPS1 and FA-A#2 IND-iPS3), FANCA expression was reconstituted as a prerequisite for generation of HIO via lentiviral expression of a doxycycline inducible construct. For radiosensitivity analysis, FANCA deficient and FANCA rescued as well as wtHIO were sham treated or irradiated with 4Gy photon, proton or carbon ions at HIT, respectively. Immunofluorescence staining of HIO for 53BP1-foci was performed 1 h post IR and gene expression analyses was performed 12 and 48 h post IR. 53BP1-foci numbers and size correlated with the higher RBE of carbon ions. A FANCA dependent differential gene expression in response to radiation was found (<i>p</i> < 0.01, ANOVA; n = 1071 12 h; n = 1100 48 h). Pathways associated with FA and DNA-damage repair i.e., transcriptional coupled nucleotide excision repair, homology-directed repair and translational synthesis were found to be differentially regulated in FANCA deficient HIO. Next, differential regulated genes were investigated as a function of radiation quality (RQ, <i>p</i> < 0.05, ANOVA; n = 742 12 h; n = 553 48 h). Interestingly, a gradual increase or decrease of gene expression was found to correlate with the three main qualities, from photon to proton and carbon irradiation. Clustering separated high-linear energy transfer irradiation with carbons from proton and photon irradiation. Genes associated with dual incision steps of TC-NER were differentially regulated in photon vs. proton and carbon irradiation. Consequently, SUMO3, ALC1, POLE4, PCBP4, MUTYH expression correlated with the higher RBE of carbon ions. An interaction between the two studied parameters FA and RQ was identified (<i>p</i> < 0.01, 2-way ANOVA n = 476). A comparison of genes regulated as a function of FA, RQ and RBE suggest a role for p53 interacting genes BRD7, EWSR1, FBXO11, FBXW8, HMGB1, MAGED2, PCBP4, and RPS27 as modulators of FA in response to radiation. This proof of concept study demonstrates that patient tailored evaluation of GI response to radiation is feasible via generation of HIO and comparative transcriptome profiling. This methodology can now be further explored for a personalized assessment of GI radiosensitivity and RBE estimation.

A novel approach for the synthesis of unsymmetrically substituted dibenzo[<i>b,f</i>][1,5]diazocine-6,12(5<i>H</i>,11<i>H</i>)diones has been developed. This facile three-step method uses variously substituted 1<i>H</i>-benzo[<i>d</i>][1,3]oxazine-2,4-diones (isatoic anhydrides) and 2-aminobenzoic acids as a starting materials. The obtained products were further transformed into <i>N</i>-alkyl-, <i>N</i>-acetyl- and dithio analogues. Developed procedures allowed the synthesis of unsymmetrical dibenzo[<i>b,f</i>][1,5]diazocine-6,12(5<i>H</i>,11<i>H</i>)diones and three novel heterocyclic scaffolds: benzo[<i>b</i>]naphtho[2,3-<i>f</i>][1,5]diazocine-6,14(5<i>H</i>,13<i>H</i>)dione, pyrido[3,2-<i>c</i>][1,5]benzodiazocine-5,11(6<i>H</i>,12<i>H</i>)-dione and pyrazino[3,2-<i>c</i>][1,5]benzodiazocine-6,12(5<i>H</i>,11<i>H</i>)dione. For 11 of the compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer (HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined. The obtained dibenzo[<i>b,f</i>][1,5]diazocine(5<i>H</i>,11<i>H</i>)6,12-dione framework could serve as a privileged structure for the drug design and development.

The formyl peptide receptor (FPR) family are a group of G-protein coupled receptors that play an important role in the regulation of inflammatory processes. It is well-established that activation of FPRs can have cardioprotective properties. Recently, more stable small-molecule FPR1/2 agonists have been described, including both Compound 17b (Cmpd17b) and Compound 43 (Cmpd43). Both agonists activate a range of signals downstream of FPR1/2 activation in human-engineered FPR-expressing cells, including ERK1/2 and Akt. Importantly, Cmpd17b (but not Cmpd43) favours bias away from intracellular Ca²⁺ mobilisation in this context, which has been associated with greater cardioprotection in response to Cmpd17b over Cmpd43. However, it is unknown whether these FPR agonists impact vascular physiology and/or elicit vasoprotective effects in the context of diabetes. First, we localized FPR1 and FPR2 receptors predominantly in vascular smooth muscle cells in the aortae of male C57BL/6 mice. We then analysed the vascular effects of Cmpd17b and Cmpd43 on the aorta using wire-myography. Cmpd17b but not Cmpd43 evoked a concentration-dependent relaxation of the mouse aorta. Removal of the endothelium or blockade of endothelium-derived relaxing factors using pharmacological inhibitors had no effect on Cmpd17b-evoked relaxation, demonstrating that its direct vasodilator actions were endothelium-independent. In aortae primed with elevated K⁺ concentration, increasing concentrations of CaCl₂ evoked concentration-dependent contraction that is abolished by Cmpd17b, suggesting the involvement of the inhibition of Ca²⁺ mobilisation via voltage-gated calcium channels. Treatment with Cmpd17b for eight weeks reversed endothelial dysfunction in STZ-induced diabetic aorta through the upregulation of vasodilator prostanoids. Our data indicate that Cmpd17b is a direct endothelium-independent vasodilator, and a vasoprotective molecule in the context of diabetes.

<h4>Aim</h4>To explore netrin-1 functions on corneal epithelium <i>in vitro</i> and <i>in vivo</i>.<h4>Methods</h4><i>In vitro</i> the human corneal epithelial (HCE) cells were treated with serum free DMEM-F12 basic media containing 0, 50, 100, 200, 300, 500, 800, and 1000 ng/mL of netrin-1, respectively. The cells viability was detected by cell counting kit-8 (CCK-8). The wound-healing assay was applied to assess the migration proficiency of HCE cells. Flow cytometry was used to analyze the cell-cycle distribution and apoptosis. <i>In vivo</i>, normal c57 (6wk) mice were demarcated with a trephine in the middle of the cornea to produce a 3-mm circular wound. Mice corneas were inflicted no epithelium with a 3-mm wound displayed, but remained the limbal epithelium intact. A blunt scalpel blade was used to remove the corneal epithelian cells, followed by topical netrin-1 application (200 ng/mL), and the group treated by PBS as control. The treated group was injected netrin-1 into the normal c57 mice inferior subconjunctival 4h before trauma. Mouse corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by TUNEL staining.<h4>Resluts</h4>A concentration of 200 ng/mL netrin-1 enhanced 25% of the HCE viability. The relative migration rates were 76.3% and 100% in control and netrin-1 treated group after cultured 72h. Treated with netrin-1 (200 ng/mL) decreased the apoptosis of HCE cells, as well as decreased their percentage from 19.3%±0.57% to 12.7%±0.42% of the total. The remaining wound area was 1.22 mm² in control group but 0.22 mm² in the netrin-1 treated group. Exogenous Netrin-1 inhibits apoptosis of corneal epithelial cells of c57 mice. TUNEL-positive cells at the epithelial layer of the corneas of the control and netrin-1 treated c57 mice at 24h after wounding were 43.3% and 16.7% respectively.<h4>Conclusion</h4>Netrin-1 can reduce HCE apoptosis as well as promote its proliferation and migration. Topical application of netrin-1 promotes the injuryed cornea epithelial wound repair and inhibits apoptosis of corneal epithelial cells. These findings may offer potential therapies to repair the defects of corneal epithelial based on netrin-1.

<h4>Background</h4>Heat stress is known to affect follicular dynamics, oocyte maturation, and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs). The present study explored the physiological and molecular response of bGCs to different heat stress intensities <i>in-vitro</i>. We exposed the primary bGCs to heat stress (HS) at 39 °C, 40 °C and 41 °C along with control samples (38 °C) for 2 h. To evaluate the impact of heat stress on bGCs, several <i>in vitro</i> cellular parameters including cell apoptosis, intracellular reactive oxygen species (ROS) accumulation and <i>HSP70</i> kinetics were assessed by flow cytometry, florescence microscopy and western blot, respectively. Furthermore, the ELISA was performed to confirm the 17β-estradiol (E₂) and progesterone (P₄) levels. In addition, the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments.<h4>Results</h4>Our findings revealed that the HS significantly decreased the cell viability, E₂ and P₄ levels in bGCs, whereas, increased the cellular apoptosis and ROS. Moreover, the RNA-Seq experiments showed that all the treatments (39 °C, 40 °C and 41 °C) significantly regulated many differentially expressed genes (DEGs) i.e. <i>BCL2L1, STAR, CYP11A1, CASP3, SOD2, HSPA13</i>, and <i>MAPK8IP1</i> and pathways associated with heat stress, apoptosis, steroidogenesis, and oxidative stress. Conclusively, our data demonstrated that the impact of 40 °C treatment was comparatively detrimental for cell viability, apoptosis and ROS accumulation. Notably, a similar trend of gene expression was reported by RT-qPCR for RNA-seq data.<h4>Conclusions</h4>Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39, 40 and 41 °C) <i>in-vitro</i>. The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle. Moreover, the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate <i>in vivo</i>. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates <i>in vitro</i>. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Deer antler is the only completely regenerable organ in mammals. During the rapid growth period, the antler proliferates even faster than cancerous tissue growth. However, the proliferation and development of antler have been in a stable and controllable growth cycle. In this study, we analyzed the time series expression data of nine samples from mesenchyme layer in three male sika deer in the early period of the antler with a saddle-like appearance (30 days), the rapid growth period of the antler with two branches (60 days), and the final period of the antler with three branches (90 days). Whole Transcriptome sequencing results show that in the 30 d versus 60 d group, 1,464 genes, 85 long noncoding RNAs (lncRNAs), and 61 miRNAs were identified as differentially expressed; 1,748 genes, 138 lncRNAs, and 78 miRNAs were identified as differentially expressed in 30d versus 90d group; and 816 differentially expressed genes (DEGs), 49 differentially expressed lncRNAs (DE lncRNAs), and 24 differentially expressed miRNA (DE miRNAs) were identified in 60d versus 90d group. A total of 182 miRNA-mRNA interaction pairs and 89 miRNA-lncRNA interaction pairs were screened from DEGs, DE miRNAs, and DE lncRNAs to construct the ceRNA regulatory network (ceRNET). In summary, we identified candidate mRNAs, miRNAs and lncRNAs that regulate the development of antler tip. It may lay the foundation for further investigating the molecular mechanism of antler rapid growth and development.

<b>Background:</b> Metabolic syndrome (MetS) is associated with the development of esophageal squamous cell carcinoma (ESCC), and long non-coding RNAs (lncRNAs) are involved in a variety of mechanisms of MetS and tumor. This study will explore the prognostic effect of MetS and the associated lncRNA signature on ESCC. <b>Methods:</b> Our previous RNA-chip data (GSE53624, GSE53622) for 179 ESCC patients were reanalyzed according to MetS. The recurrence-free survival (RFS) was collected for these patients. The status of the MetS-related tumor microenvironment was analyzed with the CIBERSORT and ESTIMATE algorithms. A lncRNA signature was established with univariate and multivariate Cox proportional hazards regression (PHR) analysis and verified using the Kaplan-Meier survival curve analysis and time-dependent receiver operating characteristic (ROC) curves. A clinical predictive model was constructed based on multiple risk factors, evaluated using C-indexes and calibration curves, and verified using data from the GEO and TCGA databases. <b>Results:</b> The results showed that MetS was an independent risk factor for ESCC patients conferring low OS and RFS. Tumor microenvironment analysis indicated that patients with MetS have high stromal scores and M2 macrophage infiltration. A six-lncRNA signature was established by 60 ESCC patients randomly selected from GSE53624 and identified with an effective predictive ability in validation cohorts (59 patients from GSE53624 and 60 patients from GSE53622), subgroup analysis, and ESCC patients from TCGA. MetS and the six-lncRNA signature could be regarded as independent risk factors and enhanced predictive ability in the clinical predictive model. <b>Conclusions:</b> Our results indicated that MetS was associated with poor prognosis in ESCC patients, and the possible mechanism was related to changes in the tumor microenvironment. MetS and the six-lncRNA signature could also serve as independent risk factors with available clinical application value.

Articular cartilage defects remain a clinical challenge. Articular cartilage defects progress to osteoarthritis, which negatively (e.g., remarkable pain, decreased mobility, distress) affects millions of people worldwide and is associated with excessive healthcare costs. Surgical procedures and cell-based therapies have failed to deliver a functional therapy. To this end, tissue engineering therapies provide a promise to deliver a functional cartilage substitute. Among the various scaffold fabrication technologies available, electrospinning is continuously gaining pace, as it can produce nano- to micro- fibrous scaffolds that imitate architectural features of native extracellular matrix supramolecular assemblies and can deliver variable cell populations and bioactive molecules. Herein, we comprehensively review advancements and shortfalls of various electrospun scaffolds in cartilage engineering.

While erythromycin has caused numerous cases of acute liver failure (ALF), clarithromycin, a similar macrolide antibiotic, has caused only six reported cases of ALF. A new case of clarithromycin-associated ALF is reported with hepatic histopathology and exclusion of other etiologies by extensive workup, and the syndrome of clarithromycin-associated ALF is better characterized by systematic review. A 60-year-old nonalcoholic man, with normal baseline liver function tests, was admitted with diffuse abdominal pain and AST = 499 U/L and ALT = 539 U/L, six days after completing a 7-day course of clarithromycin 500 mg twice daily for suspected upper respiratory infection. AST and ALT each rose to about 1,000 U/L on day-2 of admission, and rose to ≥6,000 U/L on day-3, with development of severe hepatic encephalopathy and severe coagulopathy. Planned liver biopsy was cancelled due to coagulopathies. Extensive evaluation for infectious, immunologic, and metabolic causes of liver disease was negative. Abdominal computerized tomography and abdominal ultrasound with Doppler were unremarkable. The patient developed massive, acute upper gastrointestinal bleeding associated with coagulopathies. Esophagogastroduodenoscopy was planned after massive blood product transfusions, but the patient rapidly expired from hemorrhagic shock. Autopsy revealed a golden-brown heavy liver with massive hepatic necrosis and sinusoidal congestion. Rise of AST/ALT to about 1,000 U/L each was temporally incompatible with shock liver because this rise preceded the hemorrhagic shock, but the subsequent AST/ALT rise to ≥6,000 U/L each may have had a component of shock liver. The six previously reported cases were limited by failure to exclude hepatitis E (4), lack of liver biopsy (2), and uninterpretable liver biopsy (1) and by confounding potential etiologies including disulfiram, israpidine, or recent acetaminophen use (3), clarithromycin overdose (1), active alcohol use (1), and severe heart failure (1). Review of 6 previously reported and current case of clarithromycin-associated ALF revealed that patients had AST and ALT values in the thousands. Five patients died and 2 survived.

Hepatocellular carcinoma represents one of the most aggressive cancers with high recurrence rates. The high recurrence is a major problem in the management of this disease. Cancer stem cells (CSCs) are often regarded as the basis of cancer recurrence. The anti-proliferative therapy kills the proliferating cells but induces mitotic quiescence in CSCs which remain as residual dormant CSCs. Later on, withdrawal of treatment reactivates the residual CSCs from dormancy to produce new cancer cells. The proliferation of these newly formed cancer cells initiates new tumor formation in the liver leading to tumor recurrence. HCC cells evade the immune surveillance via modulating the key immune cells by alpha feto-protein (AFP) secreted from CSCs or hepatic progenitor cells. This AFP mediated immune evasion assists in establishing new tumors by cancer cells in the liver. In this review, we will summarise the CSC mechanisms of recurrence, mitotic quiescence, dormancy and reactivation of CSCs, metastasis and immune evasion of hepatocellular carcinoma.

Combining proteomics and systems biology approaches, we demonstrate that neonatal microglial cells derived from two different CNS locations, cortex and spinal cord, and cultured <i>in vitro</i> displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited greater variability in terms of cellular and small extracellular vesicles (sEVs) protein content and levels. Bioinformatic data analysis showed that cortical microglia exerted anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia were more inflammatory. Interestingly, while both sEVs microglia sources enhanced growth of DRGs processes, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed using the neurite outgrowth assay on DRGs cells and glioma proliferation analysis in 3D spheroid cultures. Results from these <i>in vitro</i> assays suggest that the microglia localized at different CNS regions can ensure different biological functions. Together, this study indicates that neonatal microglia locations regulate their physiological and pathological functional fates and could affect the high prevalence of brain vs spinal cord gliomas in adults.

The composition of lymph nodes in pediatric patients is different from that in adults. Most notably, normal lymph nodes in children contain less macrophages. Therefore, previously described biodistributions of iron oxide nanoparticles in benign and malignant lymph nodes of adult patients may not apply to children. The purpose of our study was to evaluate if the iron supplement ferumoxytol improves the differentiation of benign and malignant lymph nodes in pediatric cancer patients on ¹⁸F-FDG PET/MRI. <b>Methods:</b> We conducted a prospective clinical trial from May 2015 to December 2018 to investigate the value of ferumoxytol nanoparticles for staging of children with cancer with ¹⁸F-FDG PET/MRI. Ferumoxytol is an FDA-approved iron supplement for the treatment of anemia and has been used "off-label" as an MRI contrast agent in this study. Forty-two children (7-18 years, 29 male, 13 female) received a ¹⁸F-FDG PET/MRI at 2 (n=20) or 24 hours (h) (n=22) after intravenous injection of ferumoxytol (dose 5 mg Fe/kg). The morphology of benign and malignant lymph nodes on ferumoxytol-enhanced T2-FSE sequences at 2 and 24 h were compared using a linear regression analysis. In addition, ADCmean-values, SUV-ratio (SUV_max lesion/SUV_mean liver) and R2*-relaxation rate of benign and malignant lymph nodes were compared with a Mann-Whitney-U test. The accuracy of different criteria was assessed with a receiver operating characteristics (ROC) curve. Follow-up imaging for at least 6 months served as the standard of reference. <b>Results:</b> We examined a total of 613 lymph nodes, of which 464 (75.7%) were benign and 149 (24.3%) were malignant. On ferumoxytol-enhanced T2-FSE images, benign lymph nodes showed a hypointense hilum and hyperintense parenchyma, while malignant lymph nodes showed no discernible hilum. This pattern was not significantly different at 2 h and 24 h postcontrast (p=0.82). Benign and malignant lymph nodes showed significantly different ferumoxytol enhancement patterns, ADCmean values of 1578 and 852 x10^-6 mm²/s, mean SUV-ratios of 0.5 and 2.8, and mean R2*-relaxation rate of 127.8 and 84.4 Hertz (Hz), respectively (all p<0.001). The accuracy of ADCmean, SUV-ratio and pattern (area under the curve (AUC): 0.99; 0.98; 0.97, respectively) was not significantly different (p=0.07). Compared to these three parameters, the accuracy of R2* was significantly lower (AUC: 0.93; p=0.001). <b>Conclusion:</b> Lymph nodes in children show different ferumoxytol-enhancement patterns on MRI than previously reported for adult patients. We found high accuracy (>90%) of ADCmean, SUV-ratio, pattern, and R2* measurements for the characterization of benign and malignant lymph nodes in children. Ferumoxytol nanoparticle accumulation at the hilum can be used to diagnose a benign lymph node. In the future, the delivery of clinically applicable nanoparticles to the hilum of benign lymph nodes could be harnessed to deliver theranostic drugs for immune cell priming.

<i><b>Purpose:</b></i> Developing chemotherapy with nanoplatforms offers a promising strategy for effective cancer treatment. In the present study, we propose a novel all-trans retinoic acid (ATRA) grafted poly beta-amino ester (PBAE) copolymer for preparing nanoparticles (NPs). <i><b>Methods:</b></i> ATRA grafted PBAE (ATRA-g-PBAE) copolymer was synthesized by grafting ATRA to PBAE; it was characterized by proton nuclear magnetic resonance, Fourier transform infrared, and thermogravimetric analysis. ATRA-g-PBAE NPs were prepared by the solvent displacement method. Design-Expert software was employed to optimize size of NPs. The morphology was evaluated by transmission electron microscope, and ultraviolet-visible spectroscopy was applied for drug release. Cytotoxicity was evaluated toward HUVEC cell line, and the 3D collagencytodex model was used to evaluate anti-angiogenic property of PBAE, ATRA, and NPs. <i><b>Results:</b></i> The optimum size of the NPs was 139.4 ± 1.41 nm. After 21 days, 66.09% ± 1.39 and 42.14% ± 1.07 of ATRA were released from NPs at pH 5.8 and 7.4, respectively. Cell culture studies demonstrated antiangiogenic effects of ATRA-g-PBAE NPs. Anti-angiogenesis IC₅₀ was 0.007 mg/mL for NPs (equal to 0.002 mg/mL of ATRA) and 0.005 mg/mL for free ATRA. <i><b>Conclusion:</b></i> This study proposes the ATRA-g-PBAE NPs with inherent anti-angiogenic effects as promising carrier for anticancer drugs with purpose of dual drug delivery.

Bone implants are susceptible to postoperative infections. Immobilization of antibiotic-loaded microparticles on implants is an effective approach to addressing this problem. Immobilization methods reported in earlier studies frequently used special or potentially harmful conditions. Therefore, the present study explored a new method to immobilize poly(lactide-<i>co</i>-glycolide) (PLGA) microspheres on bone implant materials. PLGA microspheres were prepared by an emulsion method using polyvinyl alcohol (PVA) as an emulsifier. The microspheres were immobilized on two commonly used orthopaedic biomaterials [hydroxyapatite-coated titanium (HA-Ti) and poly(methyl methacrylate) (PMMA)] by dispersing on the surface followed by vacuum drying. Microspheres were retained stably on both materials even after immersion in phosphate-buffered saline for 12 d. Pretreatment of microspheres with sodium borate (<i>i.e.</i>, an eliminator of hydroxyl groups of PVA) substantially reduced their retention on HA-Ti, but only moderately reduced their retention on PMMA. This suggested that the binding of the residual PVA on the microspheres to the HA coating is the dominant contributor to their immobilization on HA-Ti, whereas other forces contributed substantially to their immobilization on PMMA. Microspheres containing ciprofloxacin (a water-soluble antibiotic) and triclosan (an oil-soluble antibiotic) were immobilized on HA-Ti and PMMA, respectively. They effectively killed adjacent bacteria. These results offer a simple and versatile method for immobilizing drug-release microspheres on some important bone implant surfaces.

N6-methyladenosine (m⁶ A) mRNA methylation has emerged as an important player in many biological processes by regulating gene expression. However, its roles in intestinal stem cell (ISC) homeostasis remain largely unknown. Here, we report that YTHDF1, an m⁶ A reader, is highly expressed in ISCs and its expression is upregulated by Wnt signaling at the translational level. Whereas YTHDF1 is dispensable for normal intestinal development in mice, genetic ablation of Ythdf1 dramatically blocks Wnt-driven regeneration and tumorigenesis with reduced ISC stemness. Mechanistically, YTHDF1 facilitates the translation of Wnt signaling effectors including TCF7L2/TCF4, while this process is enhanced during Wnt activation to augment β-catenin activity. Targeting YTHDF1 in ISCs of established tumors leads to tumor shrinkage and prolonged survival. Collectively, our studies unveil YTHDF1 as an amplifier of Wnt/β-catenin signaling at the translational level, which is required for the maintenance of ISCs during regeneration and tumorigenesis.

<h4>Background</h4>Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral gene-delivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro.<h4>Methods</h4>First generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 × 10² infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.<h4>Results</h4>SOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels.<h4>Conclusion</h4>Adenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.

<h4>Background</h4>Influenza infections produce a spectrum of disease severity, ranging from a mild respiratory illness to respiratory failure and death. The host-response pathways associated with the progression to severe influenza disease are not well understood.<h4>Methods</h4>To gain insight into the disease mechanisms associated with progression to severe infection, we analyzed the leukocyte transcriptome in severe and moderate influenza patients and healthy control subjects. Pathway analysis on differentially expressed genes was performed using a topology-based pathway analysis tool that takes into account the interaction between multiple cellular pathways. The pathway profiles between moderate and severe influenza were then compared to delineate the biological mechanisms underpinning the progression from moderate to severe influenza.<h4>Results</h4>107 patients (44 severe and 63 moderate influenza patients) and 52 healthy control subjects were included in the study. Severe influenza was associated with upregulation in several neutrophil-related pathways, including pathways involved in neutrophil differentiation, migration, degranulation and neutrophil extracellular trap (NET) formation. The degree of upregulation in neutrophil-related pathways were significantly higher in severely infected patients compared to moderately infected patients. Severe influenza was also associated with downregulation in immune response pathways, including pathways involved in antigen presentation such as CD4+ T-cell co-stimulation, CD8+ T cell and Natural Killer (NK) cells effector functions. Apoptosis pathways were also downregulated in severe influenza patients compare to moderate and healthy controls.<h4>Conclusions</h4>These findings showed that there are changes in gene expression profile that may highlight distinct pathogenic mechanisms associated with progression from moderate to severe influenza infection.

The presence of advanced fibrosis is an important measure of the severity of chronic liver disease. Prior works that have examined the gut microbiome as a novel biomarker for advanced fibrosis have only examined patients with nonalcoholic fatty liver disease. Therefore, our goal was to examine the gut microbiome across varying etiologies of liver disease to create a predictive model for liver fibrosis based upon a microbial signature. Stool samples were obtained from patients with chronic liver disease (n = 50) undergoing FibroScan (ultrasound elastography) at the VA Greater Los Angeles Healthcare System. Healthy control patients (n = 25) were also recruited as a reference population. Fecal samples underwent 16S ribosomal RNA sequencing. Using differentially abundant microbes, a random forest classifier model was created to distinguish advanced fibrosis from mild/moderate fibrosis. The findings were then validated in a separate cohort of chronic liver disease patients (n = 37). Etiologies for liver disease included non-alcoholic liver disease (58.0%), hepatitis C (26.0%), hepatitis B (10.0%), and alcohol (6.0%). Microbiome composition was distinct in liver patients with advanced fibrosis compared to those with minimal fibrosis and healthy controls (p = 0.003). In multivariate negative binomial modeling, 26 bacterial taxa were differentially abundant in patients with advanced fibrosis as compared to those with minimal/moderate fibrosis (q-value < 0.05). A random forests classifier based on these taxa had an AUROC of 0.90 to predict advanced fibrosis. Prevotella copri, which was enriched in patients with advanced fibrosis, was the most strongly predictive microbe in the classifier. The classifier had an AUROC of 0.82 for advanced fibrosis in the validation cohort and Prevotella copri remained the strongest predictive microbe for advanced fibrosis. There is a distinct microbial signature for patients with advanced fibrosis independent of liver disease etiology and other comorbidities. These results suggest that microbial profiles can be used as a non-invasive marker for advanced fibrosis and support the hypothesis that microbes and their metabolites contribute to hepatic fibrosis.