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The measurement of Pavlovian forms of fear extinction offers a relatively simple behavioral preparation that is nonetheless tractable, from a translational perspective, as an approach to study mechanisms of exposure therapy and biological underpinnings of anxiety and trauma-related disorders such as post-traumatic stress disorder (PTSD). Deficient fear extinction is considered a robust clinical endophenotype for these disorders and, as such, has particular significance in the current "age of RDoC (research domain criteria)." Various rodent models of impaired extinction have thus been generated with the objective of approximating this clinical, relapse prone aberrant extinction learning. These models have helped to reveal neurobiological correlates of extinction circuitry failure, gene variants, and other mechanisms underlying deficient fear extinction. In addition, they are increasingly serving as tools to investigate ways to therapeutically overcome poor extinction to support long-term retention of extinction memory and thus protection against various forms of fear relapse; modeled in the laboratory by measuring spontaneous recovery, reinstatement and renewal of fear. In the current article, we review models of impaired extinction built around (1) experimentally induced brain region and neural circuit disruptions (2) spontaneously-arising and laboratory-induced genetic modifications, or (3) exposure to environmental insults, including stress, drugs of abuse, and unhealthy diet. Collectively, these models have been instrumental in advancing in our understanding of extinction failure and underlying susceptibilities at the neural, genetic, molecular, and neurochemical levels; generating renewed interest in developing novel, targeted and effective therapeutic treatments for anxiety and trauma-related disorders.

Podocyte injury is a key event for progressive renal failure. We have previously established a mouse model of inducible podocyte injury (NEP25) that progressively develops glomerulosclerosis after immunotoxin injection. We performed polysome analysis of intact and injured podocytes utilizing the NEP25 and RiboTag transgenic mice, in which a hemagglutinin tag is attached to ribosomal protein L22 selectively in podocytes. Podocyte-specific polysomes were successfully obtained by immunoprecipitation with an antihemagglutinin antibody from glomerular homogenate and analyzed using a microarray. Compared with glomerular cells, 353 genes were highly expressed and enriched in podocytes; these included important podocyte genes and also heretofore uncharacterized genes, such as Dach1 and Foxd2. Podocyte injury by immunotoxin induced many genes to be upregulated, including inflammation-related genes despite no infiltration of inflammatory cells in the glomeruli. MafF and Egr-1, which structurally have the potential to antagonize MafB and WT1, respectively, were rapidly and markedly increased in injured podocytes before MafB and WT1 were decreased. We demonstrated that Maff and Egr1 knockdown increased the MafB targets Nphs2 and Ptpro and the WT1 targets Ptpro, Nxph3, and Sulf1, respectively. This indicates that upregulated MafF and Egr-1 may promote deterioration of podocytes by antagonizing MafB and WT1. Our systematic microarray study of the heretofore undescribed behavior of podocyte genes may open new insights into the understanding of podocyte pathophysiology.

Chili peppers are an important constituent of many foods and contain medicinally valuable compounds, such as capsaicin and dihydrocapsaicin. As various dietary botanicals have anticancer properties, this study was aimed to examine the effect of Ghost pepper (Bhut Jolokia), one of the hottest chili peppers in the world, on cell proliferation, apoptosis, senescence and the global proteomic profile in human renal cell adenocarcinoma in vitro. 769-P human renal adenocarcinoma cells were cultured on RPMI-1640 media supplemented with fetal bovine serum (10%) and antibiotic-antimycotic solution (1%). Treatment stock solutions were prepared in ethanol. Cell proliferation was tested with phenol red-free media with capsaicin (0-400 μM), dihydrocapsaicin (0-400 μM), capsaicin + dihydrocapsaicin (5:1), and dry Ghost peppers (0-3 g L-1) for 24, 48 and 72 h. Polycaspase and senescence associated-beta-galactosidase (SA-beta-gal) activities were tested with capsaicin (400 μM), dihydrocapsaicin (400 μM), capsaicin (400 μM) + dihydrocapsaicin (80 μM), and ghost pepper (3 g L-1) treatments. Global proteomic profile of cells in control and ghost pepper treatment (3 g L-1) was analyzed after 6 h by a shotgun proteomic approach using tandem mass spectrometry. At 24 h after treatment (24 HAT), relative to control, cell proportion with capsaicin (400 μM), dihydrocapsaicin (400 μM), capsaicin (400 μM) + dihydrocapsaicin (80 μM), and ghost pepper (3 g L-1) treatments was reduced to 36%, 18%, 33% and 20%, respectively, and further reduced at 48 and 72 HAT. All treatments triggered an early polycaspase response. SA-beta-gal activity was normal or suppressed with all treatments. About 68,220 protein isoforms were identified by shotgun proteomic approach. Among these, about 8.2% were significantly affected by ghost pepper. Ghost pepper regulated various proteins involved in intrinsic and extrinsic apoptotic pathways, Ras, Rb/E2F, p53, TGF-beta, WNT-beta catenin, and calcium induced cell death pathways. Ghost pepper also induced changes in proteins related to methylation, acetylation, genome stability, cell cycle check points, carbohydrate, protein and other metabolism and cellular mechanisms. Ghost pepper exhibited antiproliferation activity by inducing apoptosis through a complex network of proteins in human renal cell adenocarcinoma in vitro.

Various subtypes of enteroendocrine cells (EECs) are present in the gut epithelium. EECs and pancreatic β-cells share similar pathways of differentiation during embryonic development and after birth. In this study, similarities between EECs and β-cells were evaluated in detail. To obtain specific subtypes of EECs, cell sorting by flow cytometry was conducted from STC-1 cells (a heterogenous EEC line), and each single cell was cultured and passaged. Five EEC subtypes were established according to hormone expression, measured by quantitative RT-PCR and immunostaining: L, K, I, G and S cells expressing glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, cholecystokinin, gastrin and secretin, respectively. Each EEC subtype was found to express not only the corresponding gut hormone but also other gut hormones. Global microarray gene expression profiles revealed a higher similarity between each EEC subtype and MIN6 cells (a β-cell line) than between C2C12 cells (a myoblast cell line) and MIN6 cells, and all EEC subtypes were highly similar to each other. Genes for insulin secretion-related proteins were mostly enriched in EECs. However, gene expression of transcription factors crucial in mature β-cells, such as PDX1, MAFA and NKX6.1, were remarkably low in all EEC subtypes. Each EEC subtype showed variable methylation in three cytosine-guanosine dinucleotide sites in the insulin gene (Ins2) promoter, which were fully unmethylated in MIN6 cells. In conclusion, our data confirm that five EEC subtypes are closely related to β-cells, suggesting a potential target for cell-based therapy in type 1 diabetes.

Invasive fungal infections due to <i>Candida albicans</i>, <i>Aspergillus fumigatus</i>, and <i>Cryptococcus neoformans</i> constitute a substantial threat to hospitalized immunocompromised patients. Further, the presence of drug-recalcitrant biofilms on medical devices and emergence of drug-resistant fungi, such as <i>Candida auris</i>, introduce treatment challenges with current antifungal drugs. Worse, currently there is no approved drug capable of obviating preformed biofilms, which increase the chance of infection relapses. Here, we screened a small-molecule New Prestwick Chemical Library, consisting of 1,200 FDA-approved off-patent drugs against <i>C. albicans</i>, <i>C. auris</i>, and <i>A. fumigatus</i>, to identify those that inhibit growth of all three pathogens. Inhibitors were further prioritized for their potency against other fungal pathogens and their ability to kill preformed biofilms. Our studies identified the bis-biguanide alexidine dihydrochloride (AXD) as a drug with the highest antifungal and antibiofilm activity against a diverse range of fungal pathogens. Finally, AXD significantly potentiated the efficacy of fluconazole against biofilms, displayed low mammalian cell toxicity, and eradicated biofilms growing in mouse central venous catheters <i>in vivo</i>, highlighting its potential as a pan-antifungal drug.<b>IMPORTANCE</b> The prevalence of fungal infections has seen a rise in the past decades due to advances in modern medicine leading to an expanding population of device-associated and immunocompromised patients. Furthermore, the spectrum of pathogenic fungi has changed, with the emergence of multidrug-resistant strains such as <i>C. auris</i> High mortality related to fungal infections points to major limitations of current antifungal therapy and an unmet need for new antifungal drugs. We screened a library of repurposed FDA-approved inhibitors to identify compounds with activities against a diverse range of fungi in varied phases of growth. The assays identified alexidine dihydrochloride (AXD) to have pronounced antifungal activity, including against preformed biofilms, at concentrations lower than mammalian cell toxicity. AXD potentiated the activity of fluconazole and amphotericin B against <i>Candida</i> biofilms <i>in vitro</i> and prevented biofilm growth <i>in vivo</i> Thus, AXD has the potential to be developed as a pan-antifungal, antibiofilm drug.

Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 µM) after pre-treating them with BPA (75 µM) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 µM) treated group was recovered (<i>p</i> < 0.01) by treatment with Mito-TEMPO (0.1 µM). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 µM)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.

Metal storage disorders (MSDs) are a set of rare inherited conditions with variable clinical pictures including neurological dysfunction. The objective of this study was, through a systematic review, to identify the prevalence of Parkinsonism in patients with MSDs in order to uncover novel pathways implemented in Parkinson's disease. Human studies describing patients of any age with an MSD diagnosis were analysed. Foreign language publications as well as animal and cellular studies were excluded. Searches were conducted through PubMed and Ovid between April and September 2018. A total of 53 publications were identified including 43 case reports, nine cross-sectional studies, and one cohort study. The publication year ranged from 1981 to 2018. The most frequently identified MSDs were Pantothenate kinase-associated neurodegeneration (PKAN) with 11 papers describing Parkinsonism, Hereditary hemochromatosis (HH) (7 papers), and Wilson's disease (6 papers). The mean ages of onset of Parkinsonism for these MSDs were 33, 53, and 48 years old, respectively. The Parkinsonian features described in the PKAN and HH patients were invariably atypical while the majority (4/6) of the Wilson's disease papers had a typical picture. This paper has highlighted a relationship between MSDs and Parkinsonism. However, due to the low-level evidence identified, further research is required to better define what the relationship is.

Superoxide generation by mitochondria respiratory complexes is a major source of reactive oxygen species (ROS) which are capable of initiating redox signaling and oxidative damage. Current understanding of the role of mitochondrial ROS in health and disease has been limited by the lack of experimental strategies to selectively induce mitochondrial superoxide production. The recently-developed mitochondria-targeted redox cycler MitoParaquat (MitoPQ) overcomes this limitation, and has proven effective in vitro and in Drosophila. Here we present an in vivo study of MitoPQ in the vertebrate zebrafish model in the context of Parkinson's disease (PD), and in a human cell model of Huntington's disease (HD). We show that MitoPQ is 100-fold more potent than non-targeted paraquat in both cells and in zebrafish in vivo. Treatment with MitoPQ induced a parkinsonian phenotype in zebrafish larvae, with decreased sensorimotor reflexes, spontaneous movement and brain tyrosine hydroxylase (TH) levels, without detectable effects on heart rate or atrioventricular coordination. Motor phenotypes and TH levels were partly rescued with antioxidant or monoaminergic potentiation strategies. In a HD cell model, MitoPQ promoted mutant huntingtin aggregation without increasing cell death, contrasting with the complex I inhibitor rotenone that increased death in cells expressing either wild-type or mutant huntingtin. These results show that MitoPQ is a valuable tool for cellular and in vivo studies of the role of mitochondrial superoxide generation in redox biology, and as a trigger or co-stressor to model metabolic and neurodegenerative disease phenotypes.

The placenta and decidua interact dynamically to enable embryonic and fetal development. Here, we report single-cell RNA sequencing of 14,341 and 6754 cells from first-trimester human placental villous and decidual tissues, respectively. Bioinformatic analysis identified major cell types, many known and some subtypes previously unknown in placental villi and decidual context. Further detailed analysis revealed proliferating subpopulations, enrichment of cell type-specific transcription factors, and putative intercellular communication in the fetomaternal microenvironment. This study provides a blueprint to further the understanding of the roles of these cells in the placenta and decidua for maintenance of early gestation as well as pathogenesis in pregnancy-related disorders.

<h4>Background</h4>C₆₀ fullerene-based nanoformulations are proposed to have a direct toxic effect on tumor cells. Previous investigations demonstrated that C₆₀ fullerene used alone or being conjugated with chemotherapeutic agents possesses a potent anticancer activity. The main aim of this study was to investigate the effect of C₆₀ fullerene and its nanocomplexes with anticancer drugs on human phagocyte metabolic profile in vitro.<h4>Methods</h4>Analysis of the metabolic profile of phagocytes exposed to C₆₀ fullerene in vitro revealed augmented phagocytic activity and down-regulated reactive nitrogen species generation in these cells. Additionally, cytofluorimetric analysis showed that C₆₀ fullerene can exert direct cytotoxic effect on normal and transformed phagocytes through the vigorous induction of intracellular reactive oxygen species generation.<h4>Results</h4>Cytotoxic action as well as the pro-oxidant effect of C₆₀ fullerene was more pronounced toward malignant phagocytes. At the same time, C₆₀ fullerenes have the ability to down-regulate the pro-oxidant effect of cisplatin on normal cells. These results indicate that C₆₀ fullerenes may influence phagocyte metabolism and have both pro-oxidant and antioxidant properties.<h4>Conclusions</h4>The antineoplastic effect of C₆₀ fullerene has been observed by direct toxic effect on tumor cells, as well as through the modulation of the functions of effector cells of antitumor immunity.

The aim of this study is to delineate 'admixed hybrid' and 'introgressive' Fasciola genotypes present in the Fasciola population in Vietnam. Adult liver flukes collected from ruminants in 18 Provinces were morphologically sorted out by naked eyes for small (S), medium (M) and large (L) body shapes; and human samples (n=14) from patients. Nuclear ribosomal (rDNA) ITS1 and ITS2, and mitochondrial (mtDNA) nad1 markers were used for determination of their genetic status. Total 4,725 worm samples of ruminants were tentatively classified by their size: 6% (n=284) small (S)-, 13% (n=614) medium (M)-, and 81% (n=3,827) large (L)-forms. All the representative (n=120, as 40 each group) and 14 human specimens, possessed maternal mtDNA of only F. gigantica and none of F. hepatica. Paternally, all (100%) of the L-(n=40) and 77.5% (n=31) of the M-flukes had single F. gigantica rDNA indicating 'pure' F. gigantica. A majority (90%, n=36) of the S- and 15% (n=6) of the M-worms had single F. hepatica rDNA, indicating their introgressive; the rest (10%, n=4) of the S- and 7.5% (n=3) of the M-flukes had mixture of both F. gigantica and F. hepatica rDNAs, confirming their admixed hybrid genetic status. Fourteen human samples revealed 9 (64%) of pure F. gigantica, 3 (22%) of introgressive and 2 (14%) of admixed hybrid Fasciola spp. By the present study, it was confirmed that the small worms, which are morphologically identical with F. hepatica, are admixed and/or introgressive hybrids of Fasciola spp., and able to be the pathogens of human fascioliasis.

<b>Objective:</b> Cathepsin D (CTSD) is a pivotal orchestrator in the occurrence and development of tumors. Recently, CTSD was detected in salivary adenoid cystic carcinoma (SACC). However, its functional role in perineural invasion (PNI) of SACC remained elusive. We conducted the present study to detect the expression of CTSD in SACC, analyze the correlation between CTSD expression and prognosis of SACC patients and elucidate the role of CTSD in occurrence of PNI in SACC to lay the foundation for further studies. <b>Methods:</b> Immunohistochemical analysis was conducted to assess CTSD and Ki67 expression in 158 SACC samples and 20 normal salivary gland samples adjacent to carcinoma. Meanwhile, the correlation between CTSD and PNI of SACC specimens was analyzed using Wilcoxon test. QRT-PCR, immunofluorescence and western blot analysis were used to examine the levels of CTSD mRNA and protein in SACC-LM cell line. SiRNA-mediated CTSD silence was performed. Scratch wound healing assay, transwell invasion assay and DRG co-culture assay of PNI was used to detect the ability of migration, invasion and PNI. FITC-phalloidin was used to detect cytoskeletal organization. <b>Results:</b> Our data demonstrated that the positive expression of CTSD was observed in 74.1% (117/158) of SACC cases, and the expression of CTSD was significantly correlated with the PNI (<i>p</i> < 0.05). The ability of migration, invasion, and PNI could be inhibited significantly by siRNA-mediated CTSD silence (<i>p</i> < 0.01). Furthermore, siRNA-mediated CTSD silence inhibited cytoskeletal organization and pseudo foot formation in SACC-LM cells. <b>Conclusion:</b> Our results suggested that an association between PNI and expression of CTSD existed. CTSD may promote PNI of SACC accompanied by cytoskeletal organization and pseudo foot formation.

<h4>Introduction</h4>Despite significant progress in the field of oncology, cancer remains one of the leading causes of death. Chemotherapy is one of the most common treatment options for cancer patients but is well known to result in off-target toxicity. Theranostic nanomedicines that integrate diagnostic and therapeutic functions within an all-in-one platform can increase tumor selectivity for more effective chemotherapy and aid in diagnosis and monitoring of therapeutic responses.<h4>Material and methods</h4>In this work, theranostic nanoparticles were synthesized with commonly used biocompatible and biodegradable polymers and used as cancer contrast and therapeutic agents for optical imaging and treatment of breast cancer. These core-shell nanoparticles were prepared by nanoprecipitation of blends of the biodegradable and biocompatible amphiphilic copolymers poly(lactic-<i>co</i>-glycolic acid)-<i>b</i>-poly-l-lysine and poly(lactic acid)-<i>b</i>-poly(ethylene glycol). Poly-l-lysine in the first copolymer was covalently decorated with near-infrared fluorescent Alexa Fluor 750 molecules.<h4>Results</h4>The spherical nanoparticles had an average size of 60-80 nm. The chemotherapeutic drug doxorubicin was encapsulated in the core of nanoparticles at a loading of 3% (w:w) and controllably released over a period of 30 days. A 33-fold increase in near-infrared fluorescence, mediated by protease-mediated cleavage of the Alexa Fluor 750-labeled poly-l-lysine on the surface of the nanoparticles, was observed upon interaction with the model protease trypsin. The cytocompatibility of drug-free nanoparticles and growth inhibition of drug-loaded nanoparticles on MDA-MB-231 breast cancer cells were investigated with a luminescence cell-viability assay. Drug-free nanoparticles were found to cause minimal toxicity, even at high concentrations (0.2-2,000 µg/mL), while doxorubicin-loaded nanoparticles significantly reduced cell viability at drug concentrations >10 µM. Finally, the interaction of the nanoparticles with breast cancer cells was studied utilizing fluorescence microscopy, demonstrating the potential of the nanoparticles to act as near-infrared fluorescence optical imaging agents and drug-delivery carriers.<h4>Conclusion</h4>Doxorubicin-loaded, enzymatically activatable nanoparticles of less than 100 nm were prepared successfully by nanoprecipitation of copolymer blends. These nanoparticles were found to be suitable as controlled drug delivery systems and contrast agents for imaging of cancer cells.

Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase that belongs to the casein kinase 1 family. VRK2 has long been known for its relationship with neurodegenerative disorders such as schizophrenia. However, the role of VRK2 and the substrates associated with it are unknown. Dysbindin is known as one of the strong risk factors for schizophrenia. The expression of dysbindin is indeed significantly reduced in schizophrenia patients. Moreover, dysbindin is involved in neurite outgrowth and regulation of NMDA receptor signaling. Here, we first identified dysbindin as a novel interacting protein of VRK2 through immunoprecipitation. We hypothesized that dysbindin is phosphorylated by VRK2 and further that this phosphorylation plays an important role in the function of dysbindin. We show that VRK2 phosphorylates Ser 297 and Ser 299 of dysbindin using in vitro kinase assay. In addition, we found that VRK2-mediated phosphorylation of dysbindin enhanced ubiquitination of dysbindin and consequently resulted in the decrease in its protein stability through western blotting. Over-expression of VRK2 in human neuroblastoma (SH-SY5Y) cells reduced neurite outgrowth induced by retinoic acid. Furthermore, a phosphomimetic mutant of dysbindin alleviated neurite outgrowth and affected surface expression of N-methyl-d-aspartate 2A, a subunit of NMDA receptor in mouse hippocampal neurons. Together, our work reveals the regulation of dysbindin by VRK2, providing the association of these two proteins, which are commonly implicated in schizophrenia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Dynamic assembly of macromolecules in biological systems is one of the fundamental processes that facilitates life. Although such assembly most commonly uses noncovalent interactions, a set of dynamic reactions involving reversible covalent bonding is actively being exploited for the design of functional materials, bottom-up assembly, and molecular machines. This Minireview highlights recent implementations and advancements in the area of tunable orthogonal reversible covalent (TORC) bonds for these purposes, and provides an outlook for their expansion, including the development of synthetically encoded polynucleotide mimics.

Iron, an essential element for microorganisms, functions as a vital cofactor in a wide variety of key metabolic processes. On the other hand, excess iron may have toxic effects on bacteria by catalyzing the formation of reactive oxygen species through the Fenton reaction. The prevention of iron toxicity requires the precise control of intracellular iron levels in bacteria. Mechanisms of iron homeostasis in the genus <i>Streptomyces</i> (the producers of various antibiotics) are poorly understood. <i>Streptomyces avermitilis</i> is the industrial producer of avermectins, which are potent anthelmintic agents widely used in medicine, agriculture, and animal husbandry. We investigated the regulatory role of IdeR, a DtxR family regulator, in <i>S. avermitilis</i> In the presence of iron, IdeR binds to a specific palindromic consensus sequence in promoters and regulates 14 targets involved in iron metabolism (e.g., iron acquisition, iron storage, heme metabolism, and Fe-S assembly). IdeR also directly regulates 12 targets involved in other biological processes, including morphological differentiation, secondary metabolism, carbohydrate metabolism, and the tricarboxylic acid (TCA) cycle. <i>ideR</i> transcription is positively regulated by the peroxide-sensing transcriptional regulator OxyR. A newly constructed <i>ideR</i> deletion mutant (DideR) was found to be less responsive to iron levels and more sensitive to H₂O₂ treatment than the wild-type strain, indicating that <i>ideR</i> is essential for oxidative stress responses. Our findings, taken together, demonstrate that IdeR plays a pleiotropic role in the overall coordination of metabolism in <i>Streptomyces</i> spp. in response to iron levels.<b>IMPORTANCE</b> Iron is essential to almost all organisms, but in the presence of oxygen, iron is both poorly available and potentially toxic. <i>Streptomyces</i> species are predominantly present in soil where the environment is complex and fluctuating. So far, the mechanism of iron homeostasis in <i>Streptomyces</i> spp. remains to be elucidated. Here, we characterized the regulatory role of IdeR in the avermectin-producing organism <i>S. avermitilis</i> IdeR maintains intracellular iron levels by regulating genes involved in iron absorption and storage. IdeR also directly regulates morphological differentiation, secondary metabolism, and central metabolism. <i>ideR</i> is under the positive control of OxyR and is indispensable for an efficient response to oxidative stress. This investigation uncovered that IdeR acts as a global regulator coordinating iron homeostasis, morphological differentiation, secondary metabolism, and oxidative stress response in <i>Streptomyces</i> species. Elucidation of the pleiotropic regulation function of IdeR provides new insights into the mechanisms of how <i>Streptomyces</i> spp. adapt to the complex environment.

Enantiomers of a series of tripeptide derivatives with three stereogenic centers (±)-G1-G9 have been prepared from d- and l-α-amino acids as guests for chiral recognition by ¹H NMR spectroscopy. In the meantime, a family of tetraaza macrocyclic chiral solvating agents (TAMCSAs) 1a-1d has been synthesized from d-phenylalanine and (1 S,2 S)-(+)-1,2-diaminocyclohexane. Discrimination of enantiomers of (±)-G1-G9 was carried out in the presence of TAMCSAs 1a-1d by ¹H NMR spectroscopy. The results indicate that enantiomers of (±)-G1-G9 can be effectively discriminated in the presence of TAMCSAs 1a-1d by ¹H NMR signals of multiple protons exhibiting nonequivalent chemical shifts (ΔΔδ) up to 0.616 ppm. Furthermore, enantiomers of (±)-G1-G9 were easily assigned by comparing ¹H NMR signals of the split corresponding protons with those attributed to a single enantiomer. Different optical purities (ee up to 90%) of G1 were clearly observed and calculated in the presence of TAMCSAs 1a-1d, respectively. Intermolecular hydrogen bonding interactions were demonstrated through theoretical calculations of enantiomers of (±)-G1 with TAMCSA 1a by means of the hybrid functional theory with the standard basis sets of 3-21G of the Gaussian 03 program.

<h4>Background</h4>Primary mucosal melanoma (MM) is a rare subtype of melanoma that arises from melanocytes in the mucosa. MM has not been well profiled for mutations and its etiology is not well understood, rendering current treatment strategies unsuccessful. Hence, we investigated mutational landscape for MM to understand its etiology and to clarify mutations that are potentially relevant for MM treatment.<h4>Methods</h4>Forty one MM and 48 cutaneous melanoma (CM) tissues were profiled for mutations using targeted deep next-generation sequencing (NGS) for 89 cancer-related genes. A total of 997 mutations within exons were analyzed for their mutational spectrum and prevalence of mutation, and 685 non-synonymous variants were investigated to identify mutations in individual genes and pathways. PD-L1 expression from 21 MM and 18 CM were assessed by immunohistochemistry.<h4>Results</h4>Mutational spectrum analysis revealed a lower frequency of UV-induced DNA damage in MM than in CM (p = 0.001), while tobacco exposure was indicated as a potential etiologic factor for MM. In accordance with low UV damage signatures, MM demonstrated an overall lower number of mutations compared to CM (6.5 mutations/Mb vs 14.8 mutations/Mb, p = 0.001), and less PD-L1 expression (p = 0.003). Compared to CM, which showed frequent mutations in known driver genes (BRAF 50.0%, NRAS 29.2%), MM displayed lower mutation frequencies (BRAF; 12.2%, p < 0.001, NRAS; 17.1%), and was significantly more enriched for triple wild-type (no mutations in BRAF, RAS, or NF1, 70.7% vs 25.0%, p < 0.001), IGF2R mutation (31.7% vs 6.3%, p = 0.002), and KIT mutation (9.8% vs 0%, p = 0.042). Of clinical relevance, presence of DCC mutations was significantly associated with poorer overall survival in MM (log-rank test, p = 0.02). Furthermore, mutational spectrum analysis distinguished primary anorectal MM from CM metastasized to the bowel (spectrum analysis p < 0.001, number of mutations p = 0.002).<h4>Conclusions</h4>These findings demonstrated a potential etiologic factor and driver mutation for MM and strongly suggested that MM initiation or progression involves distinct molecular-mechanisms from CM. This study also identified mutational signatures that are clinically relevant for MM treatment.

Multipotent progenitor cells confirm their T cell-lineage identity in the CD4^-CD8^- double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) belongs to the CD clan of cysteine proteases. MALT1 is a unique enzyme among this clan because it recognizes the basic amino acid arginine in the P1 pocket. Previous studies carried out with natural amino acids revealed the substrate specificity of the P4-P1 pockets of MALT1 but have provided only limited information about the catalytic preferences of this enzyme. In this study, we exploited Hybrid Combinatorial Substrate Library and Internally Quenched Fluorescence substrate technologies to interrogate the extended substrate specificity profile of the S5-S2' active site pockets using unnatural amino acids. This strategy resulted in the design of a peptide-based fluorogenic substrate, which exhibited significant activity toward MALT1. Subsequently, the substrate sequence was further utilized to develop potent, irreversible activity-based probes.

Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and β-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential β-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of β-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast β-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.

<h4>Background</h4>There has been renewal of interest in the use of prophylactic antibiotics to reduce the frequency of exacerbations and improve quality of life in chronic obstructive pulmonary disease (COPD).<h4>Objectives</h4>To determine whether or not regular (continuous, intermittent or pulsed) treatment of COPD patients with prophylactic antibiotics reduces exacerbations or affects quality of life.<h4>Search methods</h4>We searched the Cochrane Airways Group Trials Register and bibliographies of relevant studies. The latest literature search was performed on 27 July 2018.<h4>Selection criteria</h4>Randomised controlled trials (RCTs) that compared prophylactic antibiotics with placebo in patients with COPD.<h4>Data collection and analysis</h4>We used the standard Cochrane methods. Two independent review authors selected studies for inclusion, extracted data, and assessed risk of bias. We resolved discrepancies by involving a third review author.<h4>Main results</h4>We included 14 studies involving 3932 participants in this review. We identified two further studies meeting inclusion criteria but both were terminated early without providing results. All studies were published between 2001 and 2015. Nine studies were of continuous macrolide antibiotics, two studies were of intermittent antibiotic prophylaxis (three times per week) and two were of pulsed antibiotic regimens (e.g. five days every eight weeks). The final study included one continuous, one intermittent and one pulsed arm. The antibiotics investigated were azithromycin, erythromycin, clarithromycin, doxycyline, roxithromycin and moxifloxacin. The study duration varied from three months to 36 months and all used intention-to-treat analysis. Most of the pooled results were of moderate quality. The risk of bias of the included studies was generally low.The studies recruited participants with a mean age between 65 and 72 years and mostly at least moderate-severity COPD. Five studies only included participants with frequent exacerbations and two studies recruited participants requiring systemic steroids or antibiotics or both, or who were at the end stage of their disease and required oxygen. One study recruited participants with pulmonary hypertension secondary to COPD and a further study was specifically designed to asses whether eradication of Chlamydia pneumoniae reduced exacerbation rates.The co-primary outcomes for this review were the number of exacerbations and quality of life.With use of prophylactic antibiotics, the number of participants experiencing one or more exacerbations was reduced (odds ratio (OR) 0.57, 95% CI 0.42 to 0.78; participants = 2716; studies = 8; moderate-quality evidence). This represented a reduction from 61% of participants in the control group compared to 47% in the treatment group (95% CI 39% to 55%). The number needed to treat for an additional beneficial outcome with prophylactic antibiotics given for three to 12 months to prevent one person from experiencing an exacerbation (NNTB) was 8 (95% CI 5 to 17). The test for subgroup difference suggested that continuous and intermittent antibiotics may be more effective than pulsed antibiotics (P = 0.02, I² = 73.3%).The frequency of exacerbations per patient per year was also reduced with prophylactic antibiotic treatment (rate ratio 0.67; 95% CI 0.54 to 0.83; participants = 1384; studies = 5; moderate-quality evidence). Although we were unable to pool the result, six of the seven studies reporting time to first exacerbation identified an increase (i.e. benefit) with antibiotics, which was reported as statistically significant in four studies.There was a statistically significant improvement in quality of life as measured by the St George's Respiratory Questionnaire (SGRQ) with prophylactic antibiotic treatment, but this was smaller than the four unit improvement that is regarded as being clinically significant (mean difference (MD) -1.94, 95% CI -3.13 to -0.75; participants = 2237; studies = 7, high-quality evidence).Prophylactic antibiotics showed no significant effect on the secondary outcomes of frequency of hospital admissions, change in forced expiratory volume in one second (FEV1), serious adverse events or all-cause mortality (moderate-quality evidence). There was some evidence of benefit in exercise tolerance, but this was driven by a single study of lower methodological quality.The adverse events that were recorded varied among the studies depending on the antibiotics used. Azithromycin was associated with significant hearing loss in the treatment group, which was in many cases reversible or partially reversible. The moxifloxacin pulsed study reported a significantly higher number of adverse events in the treatment arm due to the marked increase in gastrointestinal adverse events (P < 0.001). Some adverse events that led to drug discontinuation, such as development of long QTc or tinnitus, were not significantly more frequent in the treatment group than the placebo group but pose important considerations in clinical practice.The development of antibiotic resistance in the community is of major concern. Six studies reported on this, but we were unable to combine results. One study found newly colonised participants to have higher rates of antibiotic resistance. Participants colonised with moxifloxacin-sensitive pseudomonas at initiation of therapy rapidly became resistant with the quinolone treatment. A further study with three active treatment arms found an increase in the degree of antibiotic resistance of isolates in all three arms after 13 weeks treatment.<h4>Authors' conclusions</h4>Use of continuous and intermittent prophylactic antibiotics results in a clinically significant benefit in reducing exacerbations in COPD patients. All studies of continuous and intermittent antibiotics used macrolides, hence the noted benefit applies only to the use of macrolide antibiotics prescribed at least three times per week. The impact of pulsed antibiotics remains uncertain and requires further research.The studies in this review included mostly participants who were frequent exacerbators with at least moderate-severity COPD. There were also older individuals with a mean age over 65 years. The results of these studies apply only to the group of participants who were studied in these studies and may not be generalisable to other groups.Because of concerns about antibiotic resistance and specific adverse effects, consideration of prophylactic antibiotic use should be mindful of the balance between benefits to individual patients and the potential harms to society created by antibiotic overuse. Monitoring of significant side effects including hearing loss, tinnitus, and long QTc in the community in this elderly patient group may require extra health resources.

Huntington's disease (HD) involves preferential and progressive degeneration of striatum and other subcortical regions as well as regional cortical atrophy. It is caused by a CAG repeat expansion in the Huntingtin gene, and the longer the expansion the earlier the age of onset. Atrophy begins prior to manifest clinical signs and symptoms, and brain atrophy in premanifest expansion carriers can be studied. We employed a diffeomorphometric pipeline to contrast subcortical structures' morphological properties in a control group with three disease groups representing different phases of premanifest HD (far, intermediate, and near to onset) as defined by the length of the CAG expansion and the participant's age (CAG-Age-Product). A total of 1,428 magnetic resonance image scans from 694 participants from the PREDICT-HD cohort were used. We found significant region-specific atrophies in all subcortical structures studied, with the estimated abnormality onset time varying from structure to structure. Heterogeneous shape abnormalities of caudate nuclei were present in premanifest HD participants estimated furthest from onset and putaminal shape abnormalities were present in participants intermediate to onset. Thalamic, hippocampal, and amygdalar shape abnormalities were present in participants nearest to onset. We assessed whether the estimated progression of subcortical pathology in premanifest HD tracked specific pathways. This is plausible for changes in basal ganglia circuits but probably not for changes in hippocampus and amygdala. The regional shape analyses conducted in this study provide useful insights into the effects of HD pathology in subcortical structures.

The mechanism underlying the role of Hsp70s in toxicity associated with intracellular accumulation of toxic protein inclusions is under intense investigation. In current study, we examined the roles of all different isoforms of yeast cytosolic Ssa Hsp70 on α-synuclein mediated cellular toxicity. The study showed that yeast cells expressing stress-inducible Ssa3 or Ssa4 as sole Ssa Hsp70 isoforms, reduced α-synuclein toxicity better than those expressing a constitutive counterpart. The protective effect of stress-inducible Ssa Hsp70s was not α-syn specific, but more general to other inclusion forming proteins such as polyQ. We show that the protective effect is not by induction of a general stress response in Ssa3 cells rather by promoting α-synuclein degradation through autophagy. The present study revealed that effect of Hsp70s was isoform dependent, and that autophagy protects Ssa3 cells from the deleterious effects of toxic protein inclusions.

<h4>Background</h4>Lung cancer is the leading cause of cancer mortality in the United States (U.S.). Squamous cell carcinoma (SQCC) represents 22.6% of all lung cancers nationally, and 26.4% in Appalachian Kentucky (AppKY), where death from lung cancer is exceptionally high. The Cancer Genome Atlas (TCGA) characterized genetic alterations in lung SQCC, but this cohort did not focus on AppKY residents.<h4>Methods</h4>Whole-exome sequencing was performed on tumor and normal DNA samples from 51 lung SQCC subjects from AppKY. Somatic genomic alterations were compared between the AppKY and TCGA SQCC cohorts.<h4>Results</h4>From this AppKY cohort, we identified an average of 237 nonsilent mutations per patient and, in comparison with TCGA, we found that <i>PCMTD1</i> (18%) and <i>IDH1</i> (12%) were more commonly altered in AppKY versus TCGA. Using <i>IDH1</i> as a starting point, we identified a mutually exclusive mutational pattern (<i>IDH1, KDM6A, KDM4E, JMJD1C</i>) involving functionally related genes. We also found actionable mutations (10%) and/or intermediate or high-tumor mutation burden (65%), indicating potential therapeutic targets in 65% of subjects.<h4>Conclusions</h4>This study has identified an increased percentage of <i>IDH1</i> and <i>PCMTD1</i> mutations in SQCC arising in the AppKY residents versus TCGA, with population-specific implications for the personalized treatment of this disease.<h4>Impact</h4>Our study is the first report to characterize genomic alterations in lung SQCC from AppKY. These findings suggest population differences in the genetics of lung SQCC between AppKY and U.S. populations, highlighting the importance of the relevant population when developing personalized treatment approaches for this disease.

The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditional <i>Ctcf</i> ablation in the MGE of mice of either sex leads to delayed tangential migration, abnormal distribution of CIN in the neocortex, a marked reduction of CINs expressing parvalbumin and somatostatin (Sst), and an increased number of MGE-derived cells expressing <i>Lhx8</i> and other markers of basal forebrain projection neurons. Likewise, <i>Ctcf</i>-null MGE cells transplanted into the cortex of wild-type hosts generate fewer Sst-expressing CINs and exhibit lamination defects that are efficiently rescued upon reexpression of LHX6. Collectively, these data indicate that CTCF regulates the dichotomy between <i>Lhx6</i> and <i>Lhx8</i> to achieve correct specification and migration of MGE-derived CINs.<b>SIGNIFICANCE STATEMENT</b> This work provides evidence that CCCTC-binding factor (CTCF) controls an early fate decision point in the generation of cortical interneurons mediated at least in part by Lhx6. Importantly, the abnormalities described could reflect early molecular and cellular events that contribute to human neurological disorders previously linked to CTCF, including schizophrenia, autism, and intellectual disability.

<h4>Introduction</h4>The common marmoset (Callithrix jacchus), a small New World monkey, has been widely used as a biological model in neuroscience to elucidate neural circuits involved in cognition and to understand brain dysfunction in neuropsychiatric disorders. In this regard, the availability of gene expression data derived from next-generation sequencing (NGS) technologies represents an opportunity for a molecular contextualization. Sexual dimorphism account for differences in diseases prevalence and prognosis. Here, we explore sex differences on frontal cortex of gene expression in common marmoset's adults.<h4>Methods</h4>Gene expression profiles in six different tissues (cerebellum, frontal cortex, liver, heart, and kidney) were analyzed in male and female marmosets. To emphasize the translational value of this species for behavioral studies, we focused on sex-biased gene expression from the frontal cortex of male and female in common marmosets and compared to humans (Homo sapiens).<h4>Results</h4>In this study, we found that frontal cortex genes whose expression is male-biased are conserved between marmosets and humans and enriched with "house-keeping" functions. On the other hand, female-biased genes are more related to neural plasticity functions involved in remodeling of synaptic circuits, stress cascades, and visual behavior. Additionally, we developed and made available an application-the CajaDB-to provide a friendly interface for genomic, expression, and alternative splicing data of marmosets together with a series of functionalities that allow the exploration of these data. CajaDB is available at cajadb.neuro.ufrn.br.<h4>Conclusion</h4>The data point to differences in gene expression of male and female common marmosets in all tissues analyzed. In frontal cortex, female-biased expression in synaptic plasticity, stress, and visual processing might be linked to biological and behavioral mechanisms of this sex. Due to the limited sample size, the data here analyzed are for exploratory purposes.

Cone snail venoms provide an ideal resource for neuropharmacological tools and drug candidates discovery, which have become a research hotspot in neuroscience and new drug development. More than 1,000,000 natural peptides are produced by cone snails, but less than 0.1% of the estimated conotoxins has been characterized to date. Hence, the discovery of novel conotoxins from the huge conotoxin resources with high-throughput and sensitive methods becomes a crucial key for the conotoxin-based drug development. In this review, we introduce the discovery methodology of new conotoxins from various <i>Conus</i> species. It focuses on obtaining full N- to C-terminal sequences, regardless of disulfide bond connectivity through crude venom purification, conotoxin precusor gene cloning, venom duct transcriptomics, venom proteomics and multi-omic methods. The protocols, advantages, disadvantages, and developments of different approaches during the last decade are summarized and the promising prospects are discussed as well.

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of <i>Onosma paniculata</i> Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. <i>β</i>,<i>β</i>-Dimethylacrylshikonin (<b>1</b>) was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than <b>1</b>. We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts. The most active compound was (<i>R</i>)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (<b>6</b>). It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2). Further investigations have shown that <b>6</b> induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.

<h4>Background</h4>Activation of the oncogene YAP has been shown to be related to lung cancer progression and associates with poor prognosis and metastasis. Metformin is a drug commonly used in the treatment of diabetes and with anticancer activity. However, the mechanism through which metformin inhibits tumorigenesis via YAP is poorly understood.<h4>Methods</h4>The mRNA and protein expressions were analyzed by RT-PCR and western blot. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of IRF-1 and 5'UTR-YAP.<h4>Findings</h4>Our immunohistochemistry staining and RT-PCR assays showed that the expression of YAP was higher in lung carcinoma samples. Interestingly, metformin was able to downregulate YAP mRNA and protein expression in lung cancer cells. Mechanistically, we found that metformin depressed YAP promoter by competing with the binding of the transcription factor IRF-1 in lung cancer cells. Moreover, combination of metformin and verteporfin synergistically inhibits cell proliferation, promotes apoptosis and suppresses cell migration/invasion by downregulating YAP, therefore reduces the side effects caused by their single use and improve the quality of life for patients with lung cancer.<h4>Interpretation</h4>we concluded that metformin depresses YAP promoter by interfering with the binding of the transcription factor IRF-1. Importantly, verteporfin sensitizes metformin-induced the depression of YAP and inhibition of cell growth and invasion in lung cancer cells. FUND: This work was supported by National Natural Science Foundation of China (No.31801085), the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017MH125), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14).

The TempO-Seq^TM platform allows for targeted transcriptomic analysis and is currently used by many groups to perform high-throughput gene expression analysis. Herein we performed a comparison of gene expression characteristics measured using 45 purified RNA samples from the livers of rats exposed to chemicals that fall into one of five modes of action (MOAs). These samples have been previously evaluated using Affymetrix^TM rat genome 230 2.0 microarrays and Illumina® whole transcriptome RNA-Seq. Comparison of these data with TempO-Seq analysis using the rat S1500+ beta gene set identified clear differences in the platforms related to signal to noise, root mean squared error, and/or sources of variability. Microarray and TempO-Seq captured the most variability in terms of MOA and chemical treatment whereas RNA-Seq had higher noise and larger differences between samples within a MOA. However, analysis of the data by hierarchical clustering, gene subnetwork connectivity and biological process representation of MOA-varying genes revealed that the samples clearly grouped by treatment as opposed to gene expression platform. Overall these findings demonstrate that the results from the TempO-Seq platform are consistent with findings on other more established approaches for measuring the genome-wide transcriptome.

<h4>Background</h4>Polycystic ovary syndrome (PCOS) is a common endocrine disorder among reproductive-aged women. While PCOS is associated with an increased risk of obesity and insulin resistance, little is known regarding the prevalence of and risk factors for nonalcoholic fatty liver disease (NAFLD) among Japanese women with PCOS. We estimated the prevalence of and risk factors for elevated liver enzymes, as the index of NAFLD, in Japanese women with PCOS.<h4>Methods</h4>We retrospectively reviewed 102 reproductive-aged women who visited the Department of Gynecology, Kyoto Medical Center in Japan from January 2000 to September 2016. Inclusion criterion was confirmed diagnosis of PCOS using International Classification of Diseases, 10th Revision, Clinical Modification (ICD-10) codes. Exclusion criteria were women with a history of liver diseases, missing body mass index (BMI) and serum alanine aminotransferase (ALT) data, and pregnancy. Data regarding age; BMI; and levels of blood glucose, serum lipid, liver enzymes, and sex hormones were obtained from medical records. Elevated liver enzymes was defined as ALT > 19 IU/L. Optimal cutoffs for risk factors for elevated liver enzymes were calculated to determine predictors of elevated liver enzymes using area under the curve (AUC) by receiver-operating characteristics (ROC).<h4>Results</h4>The prevalence of elevated liver enzymes was 33.3%. BMI was significantly higher in PCOS patients than in those without elevated liver enzymes (25.3 vs. 20.7 kg/m², P < 0.05). ROC analyses were performed using BMI and blood glucose and testosterone levels because BMI and blood glucose showed differences between the groups and testosterone is related to fatty liver. AUC of the model including BMI and blood glucose and testosterone levels was 0.861 (sensitivity, 66.7%; specificity, 100%).<h4>Conclusions</h4>These findings suggest that elevated liver enzymes are common in women with PCOS. An algorism using BMI and blood glucose and testosterone levels might be useful to determine elevated liver enzymes in women with PCOS. Our finding may be useful for the study of NAFLD among Japanese women with PCOS since several previous studies have indicated elevated liver enzymes to be related to the potential presence of NAFLD. Further examination, including abdominal ultrasonography and/or liver biopsy data, is required to confirm these results.

<h4>Background and aims</h4>In patients with nasopharyngeal carcinoma (NPC), local treatment failure and distant metastasis contribute largely to poor outcomes. The nasopharynx is an important lymphoid tissue, and NPC tumourigenesis and development are partly attributed to immune system disorders. Human leukocyte antigen F (HLA-F) has shown a close correlation with NPC in many genome-wide association studies (GWASs). However, clinical studies rarely explore the relationship of HLA-F expression with the clinical parameters and outcomes in patients with NPC.<h4>Methods</h4>In this study, we used immunohistochemistry to evaluate HLA-F expression in 74 paraffin-embedded NPC tissue sections and then analysed the association between HLA-F expression and clinical parameters and outcomes. The plasma concentration of soluble HLA-F (sHLA-F) in NPC patients and normal controls was also detected, via enzyme-linked immunosorbent assay (ELISA).<h4>Results</h4>Low, moderate, and high HLA-F expression levels were observed in 47.3% (35/74), 35.1% (26/74), and 17.6% (13/74), respectively, of the tissue samples. HLA-F expression showed a significant correlation with local recurrence (<i>p</i> = 0.037) and distant metastasis (<i>p</i> = 0.024) and was also an independent factor for local recurrence-free survival (LRFS; <i>p</i> = 0.016) and distant metastasis-free survival (DMFS; <i>p</i> = 0.004). Although the mean concentration of plasma sHLA-F in the NPC patients was higher than that in the normal controls (13.63 pg/ml vs. 10.06 pg/ml), no statistical significance was observed (<i>p</i> = 0.118).<h4>Conclusions</h4>Our study provides the first evidence that high HLA-F expression is associated with NPC local recurrence and distant metastasis and may be regarded as a poor prognostic factor for NPC patients. Additional studies using larger sample sizes may be necessary to determine whether sHLA-F is a feasible NPC diagnostic indicator.

The synergism of the electronic properties of nickel and cobalt enables bimetallic NiCo complexes to process H₂. The nickel-cobalt hydride [(dppe)Ni(pdt)(H)CoCp*]⁺ (<b>[1H]⁺</b> ) arising from protonation of the reduced state <b>1</b> was found to be an efficient electrocatalyst for H₂ evolution with Cl₂CHCOOH, and the oxidized [Ni(ii)Co(iii)]²⁺ form is capable of activating H₂ to produce <b>[1H]⁺</b> . The features of stereodynamics, acid-base properties, redox chemistry and reactivity of these bimetallic NiCo complexes in processing H₂ are potentially related to the active site of [NiFe]-H₂ases.

In Japan, methamphetamine accounts for the majority of illicit drug use and dependence is becoming a critical issue. Methamphetamine abuse induces cardiovascular complications, such as cardiomyopathy and heart failure. However, methamphetamine-associated cardiovascular complications are not common in Japan. We report the case of a young patient with hypertensive heart disease associated with habitual methamphetamine abuse. A 37-year-old man was admitted with congestive heart failure. He was a habitual methamphetamine abuser and developed chronic hypertension after he started methamphetamine abuse. His echocardiogram demonstrated left ventricular concentric hypertrophy with diffuse hypokinesis. An endomyocardial biopsy revealed histological evidence of a hypertensive heart. This case shows that habitual methamphetamine use may cause hypertensive heart disease because of chronic hypertension. <<b>Learning objective:</b> Methamphetamine-associated cardiomyopathy and congestive heart failure are uncommon in Japan. However, habitual methamphetamine abuse can be a potential cause of hypertensive heart disease due to chronic hypertension and congestive heart failure associated with hypertrophy.>.

Sjögren's syndrome is in part considered an autoimmune disease because patient sera contain antibodies binding self-structures. In fact, in addition to anti-Ro (or SSA) and anti-La (or SSB), which are included in the classification criteria, there are a wide variety of autoantibodies found among these patients. We reviewed English-language MEDLINE sources. Anti-Ro and anti-La found among healthy individuals, including mothers giving birth to infants with neonatal lupus, predicts future connective tissue disease. Those with Sjögren's syndrome can be divided into two groups; patients with only exocrine gland involvement and those with systemic disease. The presence of anti-Ro/La is associated with systemic, extraglandular disease. Rheumatoid factor is also associated with extraglandular disease while anti-cyclic citrullinated peptide (CCP) is likely associated with inflammatory arthritis and progression to rheumatoid arthritis. Anti-mitochondrial antibodies are uncommon but predict progression to primary biliary cirrhosis. Cryoglobulinemia is found in excess among those with non-Hodgkin's lymphoma. Determination of autoantibodies on the sera of Sjögren's syndrome patients has prognostic implications for Sjögren's syndrome itself as well as associated diseases.

Soybean lipoxygenase catalyzes a proton-coupled electron transfer (PCET) reaction and serves as a prototype for hydrogen tunneling in enzymes due to the unusually high kinetic isotope effect and significant modulation of the rate constant and kinetic isotope effect by mutation. Herein these experimental observations are interpreted in the context of changes to the substrate binding pocket in microsecond molecular dynamics simulations of wild-type and mutant soybean lipoxygenase. The binding pocket exhibits an hourglass shape with residues L546 and L754 bracketing the bottleneck, positioning the linoleic acid substrate for PCET. Mutation of I553 to less bulky residues slightly increases the width of the bottleneck and the volume of the binding pocket. Mutating L546 or L754 to a smaller residue also enlarges this width and volume, and mutating both has an even more dramatic effect. This analysis illustrates how mutation of the substrate binding pocket can be used as a strategy to tune the kinetics.

<h4>Background</h4>Despite their vast biological implication, the relevance of short tandem repeats (STRs)/microsatellites to the protein-coding gene translation initiation sites (TISs) remains largely unknown.<h4>Methods</h4>We performed an Ensembl-based comparative genomics study of all annotated orthologous TIS-flanking sequences in human and 46 other species across vertebrates, on the genomic DNA and cDNA platforms (755,956 TISs), aimed at identifying human-specific STRs in this interval. The collected data were used to examine the hypothesis of a link between STRs and TISs. BLAST was used to compare the initial five amino acids (excluding the initial methionine), codons of which were flanked by STRs in human, with the initial five amino acids of all annotated proteins for the orthologous genes in other vertebrates (total of 5,314,979 pair-wise TIS comparisons on the genomic DNA and cDNA platforms) in order to compare the number of events in which human-specific and non-specific STRs occurred with homologous and non-homologous TISs (i.e., ≥ 50% and < 50% similarity of the five amino acids).<h4>Results</h4>We detected differential distribution of the human-specific STRs in comparison to the overall distribution of STRs on the genomic DNA and cDNA platforms (Mann Whitney U test p = 1.4 × 10^-11 and p < 7.9 × 10^-11, respectively). We also found excess occurrence of non-homologous TISs with human-specific STRs and excess occurrence of homologous TISs with non-specific STRs on both platforms (p < 0.00001).<h4>Conclusion</h4>We propose a link between STRs and TIS selection, based on the differential co-occurrence rate of human-specific STRs with non-homologous TISs and non-specific STRs with homologous TISs.

Eukaryotic genomes are packaged into linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model <i>in vitro</i> system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of linker accessibility in the immediate vicinity of the NFR. Our results suggest that linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of linker DNA accessibility imposed by H1s.

Activation of LCK is required for canonical TCR signaling leading to T cell responses. LCK activation also initiates a negative feedback loop mediated by the phosphatase SHP-1 that turns off TCR signaling. In this article, we report that the thousand-and-one amino acid kinase 3 (TAOK3) is a key regulator of this feedback. TAOK3 is a serine/threonine kinase expressed in many different cell types including T cells. TAOK3-deficient human T cells had impaired LCK-dependent TCR signaling resulting in a defect in IL-2 response to canonical TCR signaling but not to bacterial superantigens, which use an LCK-independent pathway. This impairment was associated with enhanced interaction of LCK with SHP-1 after TCR engagement and rapid termination of TCR signals, a defect corrected by TAOK3 reconstitution. Thus, TAOK3 is a positive regulator of TCR signaling by preventing premature SHP-1-mediated inactivation of LCK. This mechanism may also regulate signaling by other Src family kinase-dependent receptors.

Determining the function of uterine lymphocytes is challenging because of the dynamic changes in response to sex hormones and, during pregnancy, to the invading foetal trophoblast cells. Here we provide a genome-wide transcriptome atlas of mouse uterine group 1 innate lymphoid cells (ILCs) at mid-gestation. Tissue-resident Eomes⁺CD49a⁺ NK cells (trNK), which resemble human uterine NK cells, are most abundant during early pregnancy, and have gene signatures associated with TGF-β responses and interactions with trophoblast, epithelial, endothelial, smooth muscle cells, leucocytes and extracellular matrix. Conventional NK cells expand late in gestation and may engage in crosstalk with trNK cells involving IL-18 and IFN-γ. Eomes^-CD49a⁺ ILC1s dominate before puberty, and specifically expand in second pregnancies when the expression of the memory cell marker CXCR6 is upregulated. These results identify trNK cells as the cellular hub of uterine group 1 ILCs, and mark CXCR6⁺ ILC1s as potential memory cells of pregnancy.

Complementary vibrational spectroscopic techniques - infrared, Raman and inelastic neutron scattering (INS) - were applied to the study of human bone burned under controlled conditions (400 to 1000 °C). This is an innovative way of tackling bone diagenesis upon burning, aiming at a quantitative evaluation of heat-induced dimensional changes allowing a reliable estimation of pre-burning skeletal dimensions. INS results allowed the concomitant observation of the hydroxyl libration (OH_libration), hydroxyl stretching (ν(OH)) and (OH_libration + ν(OH)) combination modes, leading to an unambiguous assignment of these INS features to bioapatite and confirming hydroxylation of bone's inorganic matrix. The OH_lib, ν(OH) and ν₄(PO₄^3-) bands were identified as spectral biomarkers, which displayed clear quantitative relationships with temperature revealing heat-induced changes in bone's H-bonding pattern during the burning process. These results will enable the routine use of FTIR-ATR (Fourier Transform Infrared-Attenuated Total Reflectance) for the analysis of burned skeletal remains, which will be of the utmost significance in forensic, bioanthropological and archaeological contexts.

Colonization of the upper respiratory tract by pneumococcus is important both as a determinant of disease and for transmission into the population. The immunological mechanisms that contain pneumococcus during colonization are well studied in mice but remain unclear in humans. Loss of this control of pneumococcus following infection with influenza virus is associated with secondary bacterial pneumonia. We used a human challenge model with type 6B pneumococcus to show that acquisition of pneumococcus induced early degranulation of resident neutrophils and recruitment of monocytes to the nose. Monocyte function was associated with the clearance of pneumococcus. Prior nasal infection with live attenuated influenza virus induced inflammation, impaired innate immune function and altered genome-wide nasal gene responses to the carriage of pneumococcus. Levels of the cytokine CXCL10, promoted by viral infection, at the time pneumococcus was encountered were positively associated with bacterial load.

Colorectal cancer (CRC) has a high incident rate in both men and women and is affecting millions of people every year. Genome-wide association studies (GWAS) on CRC have successfully revealed common single-nucleotide polymorphisms (SNPs) associated with CRC risk. However, they can only explain a very limited fraction of the disease heritability. One reason may be the common uni-variable analyses in GWAS where genetic variants are examined one at a time. Given the complexity of cancers, the non-additive interaction effects among multiple genetic variants have a potential of explaining the missing heritability. In this study, we employed two powerful ensemble learning algorithms, random forests and gradient boosting machine (GBM), to search for SNPs that contribute to the disease risk through non-additive gene-gene interactions. We were able to find 44 possible susceptibility SNPs that were ranked most significant by both algorithms. Out of those 44 SNPs, 29 are in coding regions. The 29 genes include <i>ARRDC5</i>, <i>DCC</i>, <i>ALK</i>, and <i>ITGA1</i>, which have been found previously associated with CRC, and <i>E2F3</i> and <i>NID2</i>, which are potentially related to CRC since they have known associations with other types of cancer. We performed pairwise and three-way interaction analysis on the 44 SNPs using information theoretical techniques and found 17 pairwise (<i>p</i> < 0.02) and 16 three-way (<i>p</i> ≤ 0.001) interactions among them. Moreover, functional enrichment analysis suggested 16 functional terms or biological pathways that may help us better understand the etiology of the disease.

Dietary iron absorption and systemic iron traffic are tightly controlled by hepcidin, a liver-derived peptide hormone. Hepcidin inhibits iron entry into plasma by binding to and inactivating the iron exporter ferroportin in target cells, such as duodenal enterocytes and tissue macrophages. Hepcidin is induced in response to increased body iron stores to inhibit further iron absorption and prevent iron overload. The mechanism involves the BMP/SMAD signaling pathway, which triggers transcriptional hepcidin induction. Inactivating mutations in components of this pathway cause hepcidin deficiency, which allows inappropriately increased iron absorption and efflux into the bloodstream. This leads to hereditary hemochromatosis (HH), a genetically heterogenous autosomal recessive disorder of iron metabolism characterized by gradual buildup of unshielded non-transferrin bound iron (NTBI) in plasma and excessive iron deposition in tissue parenchymal cells. The predominant HH form is linked to mutations in the <i>HFE</i> gene and constitutes the most frequent genetic disorder in Caucasians. Other, more severe and rare variants are caused by inactivating mutations in <i>HJV</i> (hemojuvelin), <i>HAMP</i> (hepcidin) or <i>TFR2</i> (transferrin receptor 2). Mutations in <i>SLC40A1</i> (ferroportin) that cause hepcidin resistance recapitulate the biochemical phenotype of HH. However, ferroportin-related hemochromatosis is transmitted in an autosomal dominant manner. Loss-of-function ferroportin mutations lead to ferroportin disease, characterized by iron overload in macrophages and low transferrin saturation. Aceruloplasminemia and atransferrinemia are further inherited disorders of iron overload caused by deficiency in ceruloplasmin or transferrin, the plasma ferroxidase and iron carrier, respectively.

Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against fish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine <i>ex vivo</i> and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor α (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced <i>ifn1</i> and <i>mx</i> gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches.

<h4>Background and aims</h4>Chronic hepatitis B (CHB) patients rarely achieve hepatitis B surface antigen (HBsAg) loss with nucleoside/nucleotide analog therapy.<h4>Methods</h4>In this retrospective study, it was evaluated that the rate of HBsAg loss in the HBe antigen negative (HBeAg^-) patients (n=101) treated with entecavir (ETV) for ≥24 weeks followed by switching to (n=22) or adding on (n=26) pegylated interferon (PEG-IFN), and continuing ETV (n=53).<h4>Results</h4>HBsAg clearance rate at week 48 was 9% (2/22), 15% (4/26), and 0% (0/53) (<i>P</i><0.05), in switch-to or add-on, or ETV monotherapy CHB patients, respectively. HBsAg reduction at week 48 was 1.182, 0.6614, or 0.056 log IU/mL, in switch-to, add-on, and ETV patients, respectively (<i>P</i><0.001). The response rate (HBsAg reduction >1 log IU/mL at week 48) in the switch-to, add-on, and ETV monotherapy CHB patients was 60%, 40%, and 2%, respectively (<i>P</i><0.001). In the switch-to and add-on patients, HBsAg reduction and clearance were associated with HBsAg titers at week 0 and HBsAg reduction at week 24. Furthermore, HBsAg reduction at week 24 was associated with the response rate at week 48 in the switch-to and add-on patients, showing that the area under the receiver operating characteristic curve was 0.904. Positive predictive value and negative predictive value for response rate was 70% and 100% with cut-off value 0.2 log IU/mL, respectively.<h4>Conclusion</h4>In summary, we demonstrated that PEG-IFN enhanced HBsAg loss in HBeAg^- CHB patients. High HBsAg clearance was achieved in the patients with HBsAg titers at baseline <1,000 IU/mL and HBsAg reduction >0.2 log IU/mL.

<h4>Objective</h4>There is concern regarding the safety of delayed cord clamping (DCC) in babies born at less than 34 weeks' gestation. Therefore, the primary objective of this study was to compare the rates of hyperbilirubinemia and polycythemia during initial 7 days in infants born at less than 34 weeks' gestation and randomized to receive DCC by 120 seconds or early cord clamping (ECC) within less than 30 seconds.<h4>Methods</h4>One hundred pregnant women were randomly subjected to DCC or ECC at the time of birth in a tertiary referral hospital setting. Blood samples were obtained from each newborn at 48 hours and 7 days for hematocrit measurement. Serum bilirubin levels were estimated once the infant had clinically significant jaundice or at 72 hours. For the statistical analysis, the χ² test, student's <i>t</i>-test, or Wilcoxon rank sum test was used.<h4>Results</h4>The hematocrit was significantly higher in the DCC group than in the ECC group (<i>P</i><0.001). None of the babies had polycythemia. Mean total serum bilirubin level was 6.6 mg/dL in the DCC group and 8.7 mg/dL in the ECC group (<i>P</i><0.001). There was no increased risk of hyperbilirubinemia in the DCC group.<h4>Conclusion</h4>DCC benefits preterm neonates with no significant adverse effects.

In mammals, cytosolic detection of nucleic acids is critical in initiating innate antiviral responses against invading pathogens (like bacteria, viruses, fungi and parasites). These programs are mediated by multiple cytosolic and endosomal sensors and adaptor molecules (c-GAS/STING axis and TLR9/MyD88 axis, respectively) and lead to the production of type I interferons (IFNs), pro-inflammatory cytokines, and chemokines. While the identity and role of multiple pattern recognition receptors (PRRs) have been elucidated, such immune surveillance systems must be tightly regulated to limit collateral damage and prevent aberrant responses to self- and non-self-nucleic acids. In this review, we discuss recent advances in our understanding of how cytosolic sensing of DNA is controlled during inflammatory immune responses.

Mastocytosis is a neoplastic disease originating from tissue infiltration by transformed mast cells. The diagnosis requires a high grade of suspicion due to the large variety of presenting symptoms. The World Health Organization classification recognizes localized (cutaneous) and systemic forms of the disease, with these forms showing different degrees of aggressiveness. Mastocytosis is often a multiorgan disease, and its correct management requires a multidisciplinary team of experienced consultants to provide overall patient care. Bone marrow evaluation by molecular analyses, skeleton X-ray and abdominal scan together with allergologic and dermatologic evaluation constitute the essential diagnostic work-up for adult patients with mastocytosis. As clinical situations vary, treatment options range from the use of drugs to treat the symptoms, such as anti-H1 receptors and steroids, to UV irradiation, which is overwhelmingly used in patients with cutaneous mastocytosis (CM) or indolent systemic mastocytosis, to cytoreductive treatment to control life-threatening symptoms or organ damage in the more aggressive forms of the disease. Prognosis also widely differs among patients diagnosed with mastocytosis, with the spectrum ranging from an almost normal life expectancy for those with CM and to less than 1-year median overall survival for those with mast cell leukemia.

<h4>Purpose</h4>Inherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.<h4>Design and participants</h4>We have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.<h4>Results</h4>We have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively: <i>MFN2</i>, <i>SH3TC2</i>, <i>GDAP1</i>, <i>NEFL</i>, <i>GAN</i>, <i>KIF5A</i> and <i>AARS</i>. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).<h4>Conclusion</h4>NGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.

The strict selection of pancreas for transplant has forced the development of different documents to select the suitable organ in order to minimize the risks and complications of the transplant. In 2008, Eurotransplant published the Preprocurement Pancreas Allocation Suitability Score (P-PASS) for pretransplant selection. In 2001 the Hospital Clinic of Barcelona developed a Clinical Consensus Document (CCD).<h4>Objectives</h4>We aimed to analyze the predictive decision of the pancreas acceptance to offers received in the hospital, according to the CCD criteria and compare it with the recommended value of suitability for accepting the pancreas according to the P-PASS value.<h4>Material and methods</h4>We performed a retrospective comparative study between the criteria of selection of the CCD for pancreas from 2016-2017 in comparison with the values obtained if the P-PASS had been used: ≤ 17, acceptance criteria and P-PASS; > 17, risk criteria. We defined the organ reported as rejected or accepted. The accepted organ could be procured and transplanted or discarded.<h4>Results</h4>With the CCD criteria, 7 more organs were transplanted than if we only applied the potential P-PASS criteria. In contrast, P-PASS would have ruled out an additional 9% of pancreases in relation to CCD criteria.<h4>Conclusions</h4>According our experience, it is difficult to find an adequate prediction model to select pancreas for transplantation. The application of the DCC criteria increases the number of organs valid for transplantation. At present, new criteria should be re-evaluated within multicenter studies.

Chinese hamster ovary cells, commonly used in the production of therapeutic proteins, are aneuploid. Their chromosomes bear structural abnormality and undergo changes in structure and number during cell proliferation. Some production cell lines are unstable and lose their productivity over time in the manufacturing process and during the product's life cycle. To better understand the link between genomic structural changes and productivity stability, an immunoglobulin G producing cell line was successively single-cell cloned to obtain subclones that retained or lost productivity, and their genomic features were compared. Although each subclone started with a single karyotype, the progeny quickly diversified to a population with a distribution of chromosome numbers that is not distinctive from the parent and among subclones. The comparative genomic hybridization (CGH) analysis showed that the extent of copy variation of gene coding regions among different subclones stayed at levels of a few percent. Genome regions that were prone to loss of copies, including one with a product transgene integration site, were identified in CGH. The loss of the transgene copy was accompanied by loss of transgene transcript level. Sequence analysis of the host cell and parental producing cell showed prominent structural variations within the regions prone to loss of copies. Taken together, we demonstrated the transient nature of clonal homogeneity in cell line development and the retention of a population distribution of chromosome numbers; we further demonstrated that structural variation in the transgene integration region caused cell line instability. Future cell line development may target the transgene into structurally stable regions.

The posterior cingulate cortex (PCC) and precuneus are hubs in the default mode network and play a role in processing external salient stimuli. Accordingly, activation in these regions has been associated with response to salient stimuli using drug cue-reactivity paradigms in substance using populations. These studies suggest that the PCC and precuneus may underlie deficits in processing salient stimuli that contribute toward the development of substance use disorders. The goal of this study was to directly test this hypothesis using repetitive transcranial magnetic stimulation (rTMS). Using a double-blind, placebo-controlled design, we used rTMS to target the PCC and precuneus with a double-cone coil at 10 Hz (high frequency) and 1 Hz (low frequency) in 10 adult cannabis users and 10 age- and sex-matched non-using controls. Electroencephalography data were collected before and after rTMS during a modified oddball paradigm with neutral, oddball, self-relevant, and cannabis-related stimuli. Cannabis users exhibited increased amplitude in P3 and faster latencies in the P3, N2, and P2 components in response to self-relevant stimuli compared to controls during baseline that normalized after rTMS. These results suggest that cannabis users exhibited heightened salience to external self-relevant stimuli that were modulated after rTMS. PCC dysfunction in cannabis users may be related to abnormalities in processing salient stimuli, such those during cue-reactivity, and provides a potential target for cannabis use disorder intervention.

Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (<i>nrf2</i>) target heme oxygenase 1 (<i>hmox1</i>) was upregulated in all cell lines 4 h post PTM-treatment. <i>nrf2</i> was not changed, but its baseline expression inversely and significantly correlated with <i>hmox1</i> expression after exposure to PTM. Besides awarding <i>hmox1</i> a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer. Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs. Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated. This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair.

This study aimed to assess antioxidant and immune gene transcription alterations in the gills of Sparus aurata exposed during 96 h to 4, 80, and 1600 μg/L of gold nanoparticles (AuNPs) coated with citrate or polyvinyl pyrrolidone (PVP). After 96 h of exposure, gr and cat mRNA levels decreased for all tested concentrations of AuNPs, for both coatings. Instead, gst3 mRNA increased after exposure to 1600 μg/L AuNPs (both coatings) and prdx6 increased after exposure to 1600 μg/L AuNPs-citrate. Concerning immune genes, il1β mRNA levels increased after exposure to 80 μg/L AuNPs-citrate and 1600 μg/L AuNPs-PVP and cox2 mRNA showed increased levels in fish exposed to 1600 μg/L AuNPs-citrate. Results indicate that AuNPs with distinct coatings induced different gene expression profiles in gills, though most of the studied genes remained unaltered for the tested conditions.

Prostate, bladder and kidney cancer are the three most common types of genitourinary cancer in the world. Of these, prostate and bladder cancers are within the top 10 most common cancers in men. Notably, kidney cancer causes no obvious symptoms in the early stages. To satisfy clinical-management requirements, researchers have developed numerous biomarkers by applying proteomic approaches using clinical serum, urine and tissue specimens, as well as cell and animal models. Through application of biomarker pipeline protocols, including discovery, verification and validation phases, and mass-spectrometric based proteomic platforms coupled with multiplexed quantification assays, these studies have led to recent rapid progress in this area. With improvements in mass-spectrometric based proteomic techniques, numerous promising biomarker candidates and marker panels for various clinical purposes have been proposed. Verification of novel protein biomarker candidates is very resource demanding (e.g. on the clinical and laboratory sides). With the support of national consortia, it is now possible to investigate the future clinical use of such biomarker strategies and assess their cost-effectiveness in personalized medicine.

Promoter DNA methylation, which occurs on cytosine nucleotides across CpG islands, results in gene silencing and represents a major epigenetic alteration in human cancer. Methylation-specific PCR can amplify these modifications as markers in cancer cells. In the present work, we rigorously review the published literatures describing DNA methylation in the promoters of critical tumor suppressor genes; detection of promoter DNA methylation in various body fluids permits early detection of cancer cells during perioperative courses of clinical treatment. The latest whole-genome comprehensive explorations identified excellent epigenetic biomarkers that could be detected at high frequency with high specificity; these biomarkers, which are designated highly relevant methylation genes (HRMG), permit the discrimination of tumor tissues from the corresponding normal tissues; these markers are also associated with unique cancer phenotypes, including dismal prognosis. In humans, HRMG include the CDO1, GSHR, RASSF1 and SFRP1 genes, with these markers permitting discrimination depending on the organs tested. The combination of several HRMG increased the early detection of cancer and exhibited reliable surveillance potential in human body fluids. Cancer clinics using such epigenetic biomarkers are entering a new era of enhanced decision-making with the potential for improved cancer prognosis.

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington's disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.

Microsphere beads are functionalized with oligonucleotides, antibodies, and other moieties to enable specific detection of analytes. Droplet microfluidics leverages this for single-molecule or -cell analysis by pairing beads and targets in water-in-oil droplets. Pairing is achieved with devices operating in the dripping regime, limiting throughput. Here, we describe a pairing method that uses beads to trigger the breakup of a jet into monodispersed droplets. We use the method to pair 10⁵ Human T cells with polyacrylamide beads ten times faster than methods operating in the dripping regime. Our method improves the throughput of bead-based droplet workflows, enabling analysis of large populations and the detection of rare events.

Mitochondrial dysfunction seems to play a fundamental role in the pathogenesis of neurodegeneration in Huntington's disease (HD). We assessed possible neuroprotective actions of meldonium, a small molecule affecting mitochondrial fuel metabolism, in in vitro and in vivo HD models. We found that meldonium was able to prevent cytotoxicity induced by serum deprivation, to reduce the accumulation of mutated huntingtin (mHtt) aggregates, and to upregulate the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in mHTT-expressing cells. The PGC-1α increase was accompanied by the increment of mitochondrial mass and by the rebalancing of mitochondrial dynamics with a promotion of the mitochondrial fusion. Meldonium-induced PGC-1α significantly alleviated motor dysfunction and prolonged the survival of a transgenic HD Drosophila model in which mHtt expression in the nervous system led to progressive motor performance deficits. Our study strongly suggests that PGC-1α, as a master coregulator of mitochondrial biogenesis, energy homeostasis, and antioxidant defense, is a potential therapeutic target in HD.

An essential step for understanding the transcriptional circuits that control development and physiology is the global identification and characterization of regulatory elements. Here, we present the first map of regulatory elements across the development and ageing of an animal, identifying 42,245 elements accessible in at least one <i>Caenorhabditis elegans</i> stage. Based on nuclear transcription profiles, we define 15,714 protein-coding promoters and 19,231 putative enhancers, and find that both types of element can drive orientation-independent transcription. Additionally, more than 1000 promoters produce transcripts antisense to protein coding genes, suggesting involvement in a widespread regulatory mechanism. We find that the accessibility of most elements changes during development and/or ageing and that patterns of accessibility change are linked to specific developmental or physiological processes. The map and characterization of regulatory elements across <i>C. elegans</i> life provides a platform for understanding how transcription controls development and ageing.

This study aimed to determine the impact of stocking density on the liver proteome and cecal microbiota of Peking ducks. A total of 1,200 21-day-old ducks were randomly assigned to 5 stocking density groups of 5, 6, 7, 8 and 9 ducks/m2, with 6 replicates for each group. At 40 days of age, duck serum and pectorals were collected for biochemical tests; liver and cecal contents of ducks were gathered for proteome and microbiota analysis, respectively. Serum MDA increased while pectorals T-AOC reduced linearly with enhancing stocking density. Duck lipid metabolism was altered under different stocking density as well. Serum LDL-C increased linearly with increasing stocking density. Proteome analysis revealed fatty acid biosynthesis proteins such as acyl-CoA synthetase family member 2 and fatty acid oxidation related proteins including acyl-CoA dehydrogenase long chain and acyl-coenzyme A oxidase were enriched in high stocking density group. Additionally, high stocking density increased oxidative response associated proteins such as DDRGK domain containing 1. Furthermore, increasing stocking density diminished proteins of anti-oxidant capacity including regucalcin and catalase. 16S rDNA analysis revealed that higher stocking density was accompanied with decreased microbial diversity, as well as depletion of anti-inflammatory bacterial taxa, including Bacteroidales, Butyricimonas and Alistipe. Besides, reduced bile acid metabolism-associated bacteria such as Ruminococcaceae, Clostridiales and Desulfovibrionaceae were found in the high-density group. Both proteome and 16S rDNA results showed inflammation and chronic liver disease trend in the high-density group, which suggests the involvement of the liver-gut axis in oxidative stress.

The positron emission tomography (PET) tracer [18F]MNI-659, selective for phosphodiesterase 10A (PDE10A), is a promising tool to assess an early biomarker for Huntington's disease (HD). In this study we investigated [18F]MNI-659 uptake in the Q175 mouse model of HD. Given the focal striatal distribution of PDE10A as well as the striatal atrophy occurring in HD, the spatial normalization approach applied during the processing could sensibly affect the accuracy of the regional quantification. We compared the use of a magnetic resonance images (MRI) template based on individual MRI over a PET and CT templates for regional quantification and spatial normalization of [18F]MNI-659 PET images. We performed [18F]MNI-659 PET imaging in six months old heterozygous (HET) Q175 mice and wild-type (WT) littermates, followed by X-ray computed tomography (CT) scan. In the same week, individual T2-weighted MRI were acquired. Spatial normalization and regional quantification of the PET/CT images was performed on MRI, [18F]MNI-659 PET, or CT template and compared to binding potential (BPND) using volumes manually delineated on the individual MR images. Striatal volume was significantly reduced in HET mice (-7.7%, p<0.0001) compared to WT littermates. [18F]MNI-659 BPND in striatum of HET animals was significantly reduced (p<0.0001) when compared to WT littermates using all three templates. However, BPND values were significantly higher for HET mice using the PET template compared to the MRI and CT ones (p<0.0001), with an overestimation at lower activities. On the other hand, the CT template spatial normalization introduced larger variability reducing the effect size. The PET and CT template-based approaches resulted in a lower accuracy in BPND quantification with consequent decrease in the detectability of disease effect. This study demonstrates that for [18F]MNI-659 brain PET imaging in mice the use of an MRI-based spatial normalization is recommended to achieve accurate quantification and fully exploit the detectability of disease effect.

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.

The underlining mechanisms of dietary cholesterol and nonalcoholic steatohepatitis (NASH) in contributing to hepatocellular carcinoma (HCC) remain undefined. Here we demonstrated that high-fat-non-cholesterol-fed mice developed simple steatosis, whilst high-fat-high-cholesterol-fed mice developed NASH. Moreover, dietary cholesterol induced larger and more numerous NASH-HCCs than non-cholesterol-induced steatosis-HCCs in diethylnitrosamine-treated mice. NASH-HCCs displayed significantly more aberrant gene expression-enriched signaling pathways and more non-synonymous somatic mutations than steatosis-HCCs (335 ± 84/sample vs 43 ± 13/sample). Integrated genetic and expressional alterations in NASH-HCCs affected distinct genes pertinent to five pathways: calcium, insulin, cell adhesion, axon guidance and metabolism. Some of the novel aberrant gene expression, mutations and core oncogenic pathways identified in cholesterol-associated NASH-HCCs in mice were confirmed in human NASH-HCCs, which included metabolism-related genes (ALDH18A1, CAD, CHKA, POLD4, PSPH and SQLE) and recurrently mutated genes (RYR1, MTOR, SDK1, CACNA1H and RYR2). These findings add insights into the link of cholesterol to NASH and NASH-HCC and provide potential therapeutic targets.

Outcomes for cancer patients vary greatly even within the same tumor type, and characterization of molecular subtypes of cancer holds important promise for improving prognosis and personalized treatment. This promise has motivated recent efforts to produce large amounts of multidimensional genomic (multi-omic) data, but current algorithms still face challenges in the integrated analysis of such data. Here we present Cancer Integration via Multikernel Learning (CIMLR), a new cancer subtyping method that integrates multi-omic data to reveal molecular subtypes of cancer. We apply CIMLR to multi-omic data from 36 cancer types and show significant improvements in both computational efficiency and ability to extract biologically meaningful cancer subtypes. The discovered subtypes exhibit significant differences in patient survival for 27 of 36 cancer types. Our analysis reveals integrated patterns of gene expression, methylation, point mutations, and copy number changes in multiple cancers and highlights patterns specifically associated with poor patient outcomes.

The ubiquitin specific protease, USP7, regulates multiple cellular pathways relevant for cancer through its ability to bind and sometimes stabilize specific target proteins through deubiquitylation. To gain a more complete profile of USP7 interactions in cancer cells, we performed affinity purification coupled to mass spectrometry to identify USP7 binding targets in gastric carcinoma cells. This confirmed reported associations of USP7 with USP11, PPM1G phosphatase and TRIP12 E3 ubiquitin ligase as well as identifying novel interactions with two DEAD/DEAH-box RNA helicases, DDX24 and DHX40. Using USP7 binding pocket mutants, we show that USP11, PPM1G, TRIP12 and DDX24 bind USP7 through its TRAF domain binding pocket, while DHX40 interacts with USP7 through a distinct binding pocket in the Ubl2 domain. P/A/ExxS motifs in USP11 and DDX24 that are critical for USP7 binding were also identified. Modulation of USP7 expression levels and inhibition of USP7 catalytic activity in multiple cells lines showed that USP7 consistently stabilizes DDX24, DHX40 and TRIP12 dependent on its catalytic activity, while USP11 and PPM1G levels were not consistently affected. Our study better defines the mechanisms of USP7 interaction with known targets and identifies DDX24 and DHX40 as new targets that are specifically bound and regulated by USP7.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE-MTDPS1) is a devastating autosomal recessive disorder due to mutations in <i>TYMP</i>, which cause a loss of function of thymidine phosphorylase (TP), nucleoside accumulation in plasma and tissues, and mitochondrial dysfunction. The clinical picture includes progressive gastrointestinal dysmotility, cachexia, ptosis and ophthalmoparesis, peripheral neuropathy, and diffuse leukoencephalopathy, which usually lead to death in early adulthood. Other two MNGIE-type phenotypes have been described so far, which are linked to mutations in <i>POLG</i> and <i>RRM2B</i> genes. Therapeutic options are currently available in clinical practice (allogeneic hematopoietic stem cell transplantation and carrier erythrocyte entrapped thymidine phosphorylase therapy) and newer, promising therapies are expected in the near future. Since successful treatment is strictly related to early diagnosis, it is essential that clinicians be warned about the clinical features and diagnostic procedures useful to suspect diagnosis of MNGIE-MTDPS1. The aim of this review is to promote the knowledge of the disease as well as the involved mechanisms and the diagnostic processes in order to reach an early diagnosis.

Within the subtypes of breast cancer, those identified as triple negative for expression of estrogen receptor α (ESR1), progesterone receptor (PR) and human epidermal growth factor 2 (HER2), account for 10⁻20% of breast cancers, yet result in 30% of global breast cancer-associated deaths. Thus, it is critical to develop more targeted and efficacious therapies that also demonstrate less side effects. Selenium, an essential dietary supplement, is incorporated as selenocysteine (Sec) in vivo into human selenoproteins, some of which exist as anti-oxidant enzymes and are of importance to human health. Studies have also shown that selenium compounds hinder cancer cell growth and induce apoptosis in cancer cell culture models. The focus of this study was to investigate whether selenium-antibody conjugates could be effective against triple negative breast cancer cell lines using clinically relevant, antibody therapies targeted for high expressing breast cancers and whether selenium cytotoxicity was attenuated in normal breast epithelial cells. To that end, the humanized monoclonal IgG1 antibodies, Bevacizumab and Trastuzumab were conjugated with redox selenium to form Selenobevacizumab and Selenotrastuzumab and tested against the triple negative breast cancer (TNBC) cell lines MDA-MB-468 and MDA-MB-231 as well as a normal, immortalized, human mammary epithelial cell line, HME50-5E. VEGF and HER2 protein expression were assessed by Western. Although expression levels of HER2 were low or absent in all test cells, our results showed that Selenobevacizumab and Selenotrastuzumab produced superoxide (O_2•-) anions in the presence of glutathione (GSH) and this was confirmed by a dihydroethidium (DHE) assay. Interestingly, superoxide was not elevated within HME50-5E cells assessed by DHE. The cytotoxicity of selenite and the selenium immunoconjugates towards triple negative cells compared to HME-50E cells was performed in a time and dose-dependent manner as measured by Trypan Blue exclusion, MTT assay and Annexin V assays. Selenobevacizumab and Selenotrastuzumab were shown to arrest the cancer cell growth but not the HME50-5E cells. These results suggest that selenium-induced toxicity may be effective in treating TNBC cells by exploiting different immunotherapeutic approaches potentially reducing the debilitating side effects associated with current TNBC anticancer drugs. Thus, clinically relevant, targeting antibody therapies may be repurposed for TNBC treatment by attachment of redox selenium.

Integration of multi-omics data from different molecular levels with clinical data, as well as epidemiologic risk factors, represents an accurate and promising methodology to understand the complexity of biological systems of human diseases, including cancer. By the extensive use of novel technologic platforms, a large number of multidimensional data can be derived from analysis of health and disease systems. Comprehensive analysis of multi-omics data in an integrated framework, which includes cumulative effects in the context of biological pathways, is therefore eagerly awaited. This strategy could allow the identification of pathway-addiction of cancer cells that may be amenable to therapeutic intervention. However, translation into clinical settings requires an optimized integration of omics data with clinical vision to fully exploit precision cancer medicine. We will discuss the available technical approach and more recent developments in the specific field.

<h4>Objective</h4>The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines.<h4>Methods</h4>Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay.<h4>Results</h4>Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and β2-microglobulin and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines.<h4>Conclusion</h4>These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, β2-microglobulin, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.

<b>Background:</b> Feed efficiency (FE) is an important genetic trait in poultry and livestock. Autophagy (self-eating) and proteosomes are cellular processes that remove damaged cell components (e.g., proteins, organelles). As evidence of extensive protein oxidation was observed in Pedigree Male (PedM) broilers exhibiting a low FE (LFE) phenotype compared to a high FE (HFE) phenotype, the main goal of this study was to assess gene and protein expression of the autophagy and proteosome pathways in breast muscle obtained in PedM broilers exhibiting HFE and LFE phenotypes. <b>Methods:</b> Feed efficiency was calculated as weight gain divided by feed intake gain in individual PedM broilers that were measured between 6 and 7 weeks of age. Targeted gene expression was conducted on breast muscle using quantitative real-time polymerase chain reaction (qPCR) to determine mRNA expression of genes associated with the autophagy pathway; AMP-activated protein kinase alpha 1 (AMPKα1), mammalian target of rapamycin (mTOR), Beclin 1, and autophagy genes (Atg) 3, Atg7, and Atg16L1. Binomial distribution analysis was conducted on transcriptomic and data obtained by RNAseq and shotgun proteomics, respectively on the same set of tissues for genes associated with autophagy, vacuole formation, and proteosome expression. <b>Results:</b> Greater efficiency was attained in the HFE PedM broilers by greater weight gain on the same amount of feed consumed resulting in FEs of 0.65 ± 0.01 and 0.46 ± 0.01 in the HFE and LFE phenotypes, respectively. Targeted mRNA expression analysis revealed significant (<i>P</i> < 0.05) elevations in AMPKa1, mTOR, Atg16L1, and Atg7 and a marginal (<i>P</i> = 0.07) elevation in Beclin1. Binomial distribution analysis transcriptomic and proteomic data revealed significant skews favoring autophagy-, vacuole-, and proteosome-related genes in the HFE phenotype. These results indicate that the autophagy and proteosome expression is enhanced in the HFE compared to the LFE pedigree male broiler phenotype suggesting that protein and organelle quality control may be enhanced in high feed efficiency.

Although the field of psychiatry has witnessed the proliferation of studies on Gene × Environment (G×E) interactions, still limited is the knowledge we possess of G×E interactions regarding developmental disorders. In this perspective paper, we discuss why G×E interaction studies are needed to broaden our knowledge of developmental disorders. We also discuss the different roles of hazardous versus self-generated environmental factors and how these types of factors may differentially engage with an individual's genetic background in predicting a resulting phenotype. Then, we present examplar studies that highlight the role of G×E in predicting atypical developmental trajectories as well as provide insight regarding treatment outcomes. Supported by these examples, we explore the need to move beyond merely examining statistical interactions between genes and the environment, and the motivation to investigate specific genetic susceptibility and environmental contexts that drive developmental disorders. We propose that further parsing of genetic and environmental components is required to fully understand the unique contribution of each factor to the etiology of developmental disorders. Finally, with a greater appreciation of the complexities of G×E interaction, this discussion will converge upon the potential implications for clinical and translational research.

Human hepatocellular carcinoma HepG2 cells are forced to oxidative phosphorylation (OXPHOS), when cultured in aglycemic conditions at galactose and glutamine. These Oxphos cells represent a prototype of cancer cell bioenergetics with mixed aerobic glycolysis and OXPHOS. We aimed to determine fractions of (i) glutaminolytic pathway involving aminotransferase reaction supplying 2-oxoglutarate (2OG) to the Krebs cycle vs. (ii) active segment of the Krebs cycle with aconitase and isocitrate dehydrogenase-3 (ACO-IDH3), which is typically inactive in cancer cells due to the citrate export from mitochondria. At normoxia, Oxphos cell respiration was decreased down to ~15 and ~10% by the aminotransferase inhibitor aminooxyacetate (AOA) or with AOA plus the glutamate-dehydrogenase inhibitor bithionol, respectively. Phosphorylating to non-phosphorylating respiration ratios dropped from >6.5 to 1.9 with AOA and to zero with AOA plus bithionol. Thus, normoxic Oxphos HepG2 cells rely predominantly on glutaminolysis. Addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to inhibited cells instantly partially restored respiration, evidencing the lack of 2OG-dehydrogenase substrate upon aminotransferase inhibition. Surprisingly, after 72 hr of 5% O₂ hypoxia, the AOA (bithionol) inhibition ceased and respiration was completely restored. Thus in aglycemic HepG2 cells, the hypoxia-induced factor (HIF) upregulation of glycolytic enzymes enabled acceleration of glycolysis pathway, preceded by galactolysis (Leloir pathway), redirecting pyruvate <i>via</i> still incompletely blocked pyruvate dehydrogenase toward the ACO-IDH3. Glycolytic flux upregulation at hypoxia was evidently matched by a higher activity of the Leloir pathway in Oxphos cells. Hypoxic Oxphos cells increased 2-fold the NADPH oxidase activity, whereas hypoxic glycolytic cells decreased it. Oxphos cells and glycolytic cells at 5 mM glucose decreased their reduced glutathione fraction. In contrast to aglycemic cells, glycolytic HepG2 cells decreased their respiration at hypoxia despite the dm2OG presence, i.e., even at unlimited respiratory substrate availability for 72 hr at 5% O₂, exhibiting the canonical HIF-mediated adaptation. Nevertheless, their ATP content was much higher with dm2OG as compared to its absence during hypoxic adaptation. Thus, the metabolic plasticity of cancer cells is illustrated under conditions frequently established for solid tumors <i>in vivo</i>, such as aglycemia plus hypoxia. Consequently, a wide acceptance of the irreversible and exclusive Warburg phenotype in cancer cells is incorrect.

HER3, a member of the receptor tyrosine kinase super family, is overexpressed in a number of cancers, and is associated with malignant phenotypes. Control of the protein stability of the membrane, as well as nuclear receptors, has been known to be an important process affecting tumor cells; however, their relationships have yet to be elucidated. In this study, we demonstrate that estradiol promotes rapid degradation of HER3 via the proteasome pathway in ER-positive breast cancer, MCF-7. ER prevented HER3 degradation, and knockdown of ER expression by si-RNA promoted rapid degradation of HER3. Breakdown of HER3 and ER were regulated by a ubiquitin ligase Nedd4-1 in the presence of estradiol stimulation. We speculate that estradiol quickly degrades ER, making HER3 accessible by Nedd4-1, and leads to the rapid degradation of HER3. In addition, knockdown of ubiquitin ligase Nedd4-1 enhances estradiol induced cell proliferation. These results indicate that HER3 and Nedd4-1 in ER-positive breast cancers might be an important therapeutic target.

The discipline of neurotheranostics was forged to improve diagnostic and therapeutic clinical outcomes for neurological disorders. Research was facilitated, in largest measure, by the creation of pharmacologically effective multimodal pharmaceutical formulations. Deployment of neurotheranostic agents could revolutionize staging and improve nervous system disease therapeutic outcomes. However, obstacles in formulation design, drug loading and payload delivery still remain. These will certainly be aided by multidisciplinary basic research and clinical teams with pharmacology, nanotechnology, neuroscience and pharmaceutic expertise. When successful the end results will provide "optimal" therapeutic delivery platforms. The current report reviews an extensive body of knowledge of the natural history, epidemiology, pathogenesis and therapeutics of neurologic disease with an eye on how, when and under what circumstances neurotheranostics will soon be used as personalized medicines for a broad range of neurodegenerative, neuroinflammatory and neuroinfectious diseases.

Huntington's disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca²⁺ release from the endoplasmic reticulum (ER) induces Ca²⁺ influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca²⁺ depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IP₃R1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IP₃R1 was detected while the ionomycin-sensitive ER Ca²⁺ pool decreased. 6-Bromo-<i>N</i>-(2-phenylethyl)-2,3,4,9-tetrahydro-1<i>H</i>-carbazol-1-amine hydrochloride (C₂₀H₂₂BrClN₂), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP₃ sponge also restored the elevation of SOCE and increased the release of Ca²⁺ from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IP₃R1 activity.

No abstract available.

Long noncoding RNA FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) dysregulation associates with multiple types of human cancer. However, the biological functions of FENDRR in renal cell carcinoma are unresolved. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression level of FENDRR in renal cell carcinoma tissues. An RNA interference assay and ectopic expression experiments were conducted to evaluate the effects of FENDRR on cell proliferation, migration, invasion and colony formation <i>in vitro</i>. RNA immunoprecipitation was conducted to identify proteins associated with FENDRR. It was observed that FENDRR is frequently downregulated in renal cell carcinoma and overexpression of FENDRR attenuated proliferation, migration, invasion and colony growth of renal carcinoma cells. Conversely, knockdown of FENDRR promotes proliferation and invasiveness of renal carcinoma cells. Downregulation of FENDRR associates with poor prognosis of renal cell carcinoma. Mechanistically, it was identified that FENDRR may bind to Polycomb Repressive Complex 2 and lysin methyltransferase 2A histone modifying complexes. In summary, FENDRR acts as an tumor suppressor in renal cell carcinoma and may serve as a candidate target for gene therapy.

Regenerative therapies for bone and cartilage injuries are currently unable to replicate the complex microenvironment of native tissue. There are many tissue engineering approaches attempting to address this issue through the use of synthetic materials. Although synthetic materials can be modified to simulate the mechanical and biochemical properties of the cell microenvironment, they do not mimic in full the multitude of interactions that take place within tissue. Decellularized extracellular matrix (dECM) has been established as a biomaterial that preserves a tissue's native environment, promotes cell proliferation, and provides cues for cell differentiation. The potential of dECM as a therapeutic agent is rising, but there are many limitations of dECM restricting its use. This review discusses the recent progress in the utilization of bone and cartilage dECM through applications as scaffolds, particles, and supplementary factors in bone and cartilage tissue engineering.

The design and synthesis of a series of novel d-(+)-camphor <i>N</i>-acylhydrazones exhibiting inhibitory activity against vaccinia and influenza viruses are presented. An easy pathway to camphor-based <i>N</i>-acylhydrazones containing in their structure aliphatic, aromatic, and heterocyclic pharmacophore scaffolds has been developed. The conformation and configuration of the synthesized hydrazones were thoroughly characterized by a complete set of spectral characterization techniques, including 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis. <i>In vitro</i> screening for activity against vaccinia virus (VV) and influenza H1N1 virus was carried out for the obtained compounds. It was revealed that the derived <i>N</i>-acylhydrazones exhibited significant antiviral activity with a selectivity index >280 against VV for the most promising compound.

<b><i>Background and Aims:</i></b> Although hypovitaminosis D is common among patients with chronic liver disease (CLD), the data are inconsistent on its prevalence and its relationship with CLD. This study aimed to estimate the prevalence of hypovitaminosis D among patients with CLD and to determine the relationship between hypovitaminosis D and severity of liver dysfunction, and calcium (Ca), phosphate (PO4) and parathyroid hormone (PTH) levels in CLD. <b><i>Methods:</i></b> The study included 236 CLD patients attending the Department of Hepatology, Rajiv Gandhi Government General Hospital (Chennai, India). Serum levels of 25-hydroxyvitamin D (25(OH)D), PTH, Ca, and PO4 were estimated. Severity of liver dysfunction was graded using the Child-Turcotte-Pugh (CTP) score. <b><i>Results:</i></b> The first report from our population showed that 162 of 236 (68.6%) CLD patients had hypovitaminosis D (25(OH)D levels of <30 ng/mL), with higher frequency (124/162) 76.5% among CTP B, C patients. Significant negative correlation (<i>r</i> = -0.288, <i>p</i> = 0.0001) between 25(OH)D and CTP scores was noted in hypovitaminosis D conditions. Level of 25(OH)D was correlated negatively with PTH (<i>r</i> = -0.537, <i>p</i> = 0.0001), positively with Ca (<i>r</i> = 0.657, <i>p</i> = 0.0001), and positively with PO4 (<i>r</i> = 0.477, <i>p</i> = 0.0001) in sufficient vitamin D conditions. <b><i>Conclusions:</i></b> Hypovitaminosis D is associated with higher CTP scores and is strongly associated with dysregulation of the Ca-PTH-vitamin D axis in CLD. Timely measurement of vitamin D levels is essential, along with levels of PTH, Ca and PO4, to manage CLD patients.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). Causative genetic alterations in MF are unknown. The low recurrence of pathogenic small-scale mutations (ie, nucleotide substitutions, indels) in the disease, calls for the study of additional aspects of MF genetics. Here, we investigated structural genomic alterations in tumor-stage MF by integrating whole-genome sequencing and RNA-sequencing. Multiple genes with roles in cell physiology (n = 113) and metabolism (n = 92) were found to be impacted by genomic rearrangements, including 47 genes currently implicated in cancer. Fusion transcripts involving genes of interest such as DOT1L, KDM6A, LIFR, TP53, and TP63 were also observed. Additionally, we identified recurrent deletions of genes involved in cell cycle control, chromatin regulation, the JAK-STAT pathway, and the PI-3-K pathway. Remarkably, many of these deletions result from genomic rearrangements. Deletion of tumor suppressors HNRNPK and SOCS1 were the most frequent genetic alterations in MF after deletion of CDKN2A. Notably, SOCS1 deletion could be detected in early-stage MF. In agreement with the observed genomic alterations, transcriptome analysis revealed up-regulation of the cell cycle, JAK-STAT, PI-3-K and developmental pathways. Our results position inactivation of HNRNPK and SOCS1 as potential driver events in MF development.

For patients with incurable neurodegenerative disorders such as Huntington's (HD) and Parkinson's disease, cell transplantation has been explored as a potential treatment option. Here, we present the first clinicopathological study of a patient with HD in receipt of cell-suspension striatal allografts who took part in the NEST-UK multicenter clinical transplantation trial. Using various immunohistochemical techniques, we found a discrepancy in the survival of grafted projection neurons with respect to grafted interneurons as well as major ongoing inflammatory and immune responses to the grafted tissue with evidence of mutant huntingtin aggregates within the transplant area. Our results indicate that grafts can survive more than a decade post-transplantation, but show compromised survival with inflammation and mutant protein being observed within the transplant site. Ann Neurol 2018;84:950-956.

<h4>Background</h4>Diabetic peripheral neurovascular diseases (DPNVs) are complex, lacking effective treatment. Autologous/allogeneic transplantation of adipose-derived stem cells (ADSCs) is a promising strategy for DPNVs. Nonetheless, the transplanted ADSCs demonstrate unsatisfying viability, migration, adhesion, and differentiation in vivo, which reduce the treatment efficiency. Netrin-1 secreted as an axon guidance molecule and served as an angiogenic factor, demonstrating its ability in enhancing cell proliferation, migration, adhesion, and neovascularization.<h4>Methods</h4>ADSCs acquired from adipose tissue were modified by Netrin-1 gene (NTN-1) using the adenovirus method (N-ADSCs) and proliferation, migration, adhesion, and apoptosis examined under high-glucose condition. The sciatic denervated mice (db/db) with type 2 diabetes mellitus (T2DM) were transplanted with N-ADSCs and treatment efficiency assessed based on the laser Doppler perfusion index, immunofluorescence, and histopathological assay. Also, the molecular mechanisms underlying Netrin-1-mediated proliferation, migration, adhesion, differentiation, proangiogenic capacity, and apoptosis of ADSCs were explored.<h4>Results</h4>N-ADSCs improved the proliferation, migration, and adhesion and inhibited the apoptosis of ADSCs in vitro in the condition of high glucose. The N-ADSCs group demonstrated an elevated laser Doppler perfusion index in the ADSCs and control groups. N-ADSCs analyzed by immunofluorescence and histopathological staining demonstrated the distribution of the cells in the injected limb muscles, indicating chronic ischemia; capillaries and endothelium were formed by differentiation of N-ADSCs. The N-ADSCs group showed a significantly high density of the microvessels than the ADSCs group. The upregulation of AKT/PI3K/eNOS/P-38/NF-κB signaling pathways and secretion of multiple growth factors might explain the positive effects of Netrin-1 on ADSCs.<h4>Conclusion</h4>The overexpression of Netrin-1 in ADSCs improves proliferation, migration, and treatment effect in type 2 diabetic mice with sciatic denervation, which directs the clinical treatment of patients with DPNVs.

<h4>Background</h4>Human decidua basalis mesenchymal stem/multipotent stromal cells (DBMSCs) inhibit endothelial cell activation by inflammation induced by monocytes. This property makes them a promising candidate for cell-based therapy to treat inflammatory diseases, such as atherosclerosis. This study was performed to examine the ability of DBMSCs to protect endothelial cell functions from the damaging effects resulting from exposure to oxidatively stress environment induced by H₂O₂ and monocytes.<h4>Methods</h4>DBMSCs were co-cultured with endothelial cells isolated from human umbilical cord veins in the presence of H₂O₂ and monocytes, and various functions of endothelial cell were then determined. The effect of DBMSCs on monocyte adhesion to endothelial cells in the presence of H₂O₂ was also examined. In addition, the effect of DBMSCs on HUVEC gene expression under the influence of H₂O₂ was also determined.<h4>Results</h4>DBMSCs reversed the effect of H₂O₂ on endothelial cell functions. In addition, DBMSCs reduced monocyte adhesion to endothelial cells and also reduced the stimulatory effect of monocytes on endothelial cell proliferation in the presence of H₂O₂. Moreover, DBMSCs modified the expression of many genes mediating important endothelial cell functions. Finally, DBMSCs increased the activities of glutathione and thioredoxin reductases in H₂O₂-treated endothelial cells.<h4>Conclusions</h4>We conclude that DBMSCs have potential for therapeutic application in inflammatory diseases, such as atherosclerosis by protecting endothelial cells from oxidative stress damage. However, more studies are needed to elucidate this further.

Paracoccidioidomycosis (PCM) is a systemic disease caused by thermodymorphic fungi of the Paracoccidioides brasiliensis complex, (Paracoccidioides spp.). Patients with PCM reveal specific cellular immune impairment. Despite the effective treatment, quiescent fungi can lead to relapse, usually late, the serological diagnosis of which has been deficient. The present study was carried out with the objective of investigating a biomarker for the identification of PCM relapse and another molecule behaving as an immunological recovery biomarker; therefore, it may be used as a cure criterion. In the evolutionary analysis of the proteins identified in PCM patients, comparing those that presented with those that did not reveal relapse, 29 proteins were identified. The interactions observed between the proteins, using transferrin and haptoglobin, as the main binding protein, were strong with all the others. Patient follow-up suggests that cerulosplamin may be a marker of relapse and that transferrin and apolipoprotein A-II may contribute to the evaluation of the treatment efficacy and avoiding a premature decision.

<h4>Background</h4>In response to a wound, fibroblasts are activated to migrate toward the wound, to proliferate and to contribute to the wound healing process. We hypothesize that changes in pre-mRNA processing occurring as fibroblasts enter the proliferative cell cycle are also important for promoting their migration.<h4>Results</h4>RNA sequencing of fibroblasts induced into quiescence by contact inhibition reveals downregulation of genes involved in mRNA processing, including splicing and cleavage and polyadenylation factors. These genes also show differential exon use, especially increased intron retention in quiescent fibroblasts compared to proliferating fibroblasts. Mapping the 3' ends of transcripts reveals that longer transcripts from distal polyadenylation sites are more prevalent in quiescent fibroblasts and are associated with increased expression and transcript stabilization based on genome-wide transcript decay analysis. Analysis of dermal excisional wounds in mice reveals that proliferating cells adjacent to wounds express higher levels of cleavage and polyadenylation factors than quiescent fibroblasts in unwounded skin. Quiescent fibroblasts contain reduced levels of the cleavage and polyadenylation factor CstF-64. CstF-64 knockdown recapitulates changes in isoform selection and gene expression associated with quiescence, and results in slower migration.<h4>Conclusions</h4>Our findings support cleavage and polyadenylation factors as a link between cellular proliferation state and migration.

<h4>Background</h4>With the rapid integration of genetics into medicine, it has become evident that practicing physicians as well as medical students and clinical researchers need to be updated on the fundamentals of bioinformatics. To achieve this, the following gaps need to be addressed: a lack of defined learning objectives for "Bioinformatics for Medical Practitioner" courses, an absence of a structured lesson plan to disseminate the learning objectives, and no defined step-by-step strategy to teach the essentials of bioinformatics in the medical curriculum.<h4>Objective</h4>The objective of this study was to address these gaps to design a streamlined pedagogical strategy for teaching basics of bioinformatics in the undergraduate medical curriculum.<h4>Methods</h4>The established instructional design strategies employed in medical education-Gagne's 9 events of instruction-were followed with further contributions from Peyton's four-step approach to design a structured lesson plan in bioinformatics.<h4>Results</h4>First, we defined the specifics of bioinformatics that a medical student or health care professional should be introduced to use this knowledge in a clinical context. Second, we designed a structured lesson plan using a blended approach from both Gagne's and Peyton's instructional models. Lastly, we delineated a step-by-step strategy employing free Web-based bioinformatics module, combining it with a clinical scenario of familial hypercholesterolemia to disseminate the defined specifics of bioinformatics. Implementation of Schon's reflective practice model indicated that the activity was stimulating for the students with favorable outcomes regarding their basic training in bioinformatics.<h4>Conclusions</h4>To the best of our knowledge, the present lesson plan is the first that outlines an effective dissemination strategy for integrating introductory bioinformatics into a medical curriculum. Further, the lesson plan blueprint can be used to develop similar skills in workshops, continuing professional development, or continuing medical education events to introduce bioinformatics to practicing physicians.

Condensin is a multi-subunit protein complex that belongs to the family of structural maintenance of chromosomes (SMC) complexes. Condensins regulate chromosome structure in a wide range of processes including chromosome segregation, gene regulation, DNA repair and recombination. Recent research defined the structural features and molecular activities of condensins, but it is unclear how these activities are connected to the multitude of phenotypes and functions attributed to condensins. In this review, we briefly discuss the different molecular mechanisms by which condensins may regulate global chromosome compaction, organization of topologically associated domains, clustering of specific loci such as tRNA genes, rDNA segregation, and gene regulation.

Ultraviolet (UV) radiation induces skin photoaging, which is associated with the elevation of matrix metalloproteinases (MMPs) and the impairment of collagen. The Euphrasia species play a well-known role in the treatment of certain eye disorders through their anti-oxidative and anti-inflammatory activities. However, their protective activity toward UVB-induced damage remains unclear. In the present study, we investigated the protective effect of <i>Euphrasia officinalis</i> (95% ethanol extract) on UVB-irradiated photoaging in normal human dermal fibroblasts (NHDFs). Our results show that <i>Euphrasia officinalis</i> extract exhibited obvious reactive oxygen species (ROS) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, enhanced NHDF cell migration, and reduced UVB-induced apoptosis. The UVB-induced increases in MMP-1 and MMP-3 and decrease in type I procollagen were ameliorated by <i>Euphrasia officinalis</i> treatment, which worked by suppressing the mitogen-activated protein kinase (MAPK) and nuclear transcription factor activator protein 1 (AP-1) signaling pathways. Taken together, our data strongly suggest that <i>Euphrasia officinalis</i> ethanol extract could reduce UVB-induced photoaging by alleviating oxidative stress, proinflammatory activity, and cell apoptosis.

Retinal vein occlusion (RVO) is a common retinal vascular disease. RVO may be complicated by pronounced ischemia that often leads to severe loss of visual function. The present work aimed at studying the retinal proteome of RVO complicated by ischemia. In six Danish Landrace pigs RVO was induced with argon laser in the right eye of each animal. As four retinal veins were occluded, the RVO best corresponded to a central retinal vein occlusion (CRVO). Left control eyes received a similar laser treatment without inducing occlusion. RVO and retinal ischemia were verified by angiography. The retinas were collected 15 days after RVO for proteomic analysis. RVO resulted in a downregulation of proteins involved in visual perception, including rhodopsin, transducin alpha chain, and peripherin-2. RVO also caused a downregulation of proteins involved in neurotransmitter transport, including glutamate decarboxylase 1 (GAD1), glutamate decarboxylase 2 (GAD2), and complexins 2⁻4. RVO lead to increased contents of proteins involved in inflammation, including interleukin-18 (IL-18), S100A12, and annexin A1 (ANXA1). Immunohistochemistry revealed a general retinal upregulation of IL-18 and ANXA1 while S100A12 was highly abundant in retinal ganglion cells in RVO. IL-18 and S100A12 are likely to be driving forces in the inflammatory response of RVO complicated by ischemia. Our findings also suggest that RVO results in compromised neurotransmission and a downregulation of proteins involved in visual perception.

<h4>Introduction</h4>Altered levels of factor (F)VIII, prothrombin, or antithrombin have been associated with an increased risk for venous thromboembolism (VTE). However, the exact molecular mechanism by which these altered factor levels modulate the risk is incompletely understood. Here we hypothesize that elevated factor levels affect the pro- and anticoagulant balance in coagulation such that even minute amounts of tissue factor (TF) will initiate thrombin formation, thereby contributing to the VTE risk.<h4>Materials and methods</h4>To test this so-called TF-threshold hypothesis, we monitored thrombin generation initiated by very low TF concentrations in FXII-deficient plasma, to avoid any contact pathway-mediated thrombin formation. Furthermore, similar experiments were performed in the presence of increasing concentrations of pro- and anticoagulant proteins.<h4>Results</h4>A TF-threshold was established in the FXII-deficient plasma, which is subject to inter-individual variation. Elevated plasma levels of procoagulant factors, such as FVIII or prothrombin, enhanced thrombin generation and reduced the amount of TF required for the initiation of thrombin formation. Conversely, elevated levels of the coagulation inhibitor antithrombin increased the TF-threshold.<h4>Conclusions</h4>Our findings support a mediating role for the TF-threshold in the association between high procoagulant factor levels and the risk for VTE. Furthermore, elevated levels of anticoagulants may have a protective effect on the development of VTE.

Follicular helper T cells (Tfh) are specialized helper T cells that are predominantly located in germinal centers and provide help to B cells. The development and differentiation of Tfh cells has been shown to be regulated by transcription factors, such as B-cell lymphoma 6 protein (Bcl-6), signal transducer and activator of transcription 3 (STAT3) and B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, cytokines, including IL-21, have been found to be important for Tfh cell development. Moreover, several epigenetic modifications have also been reported to be involved in the determination of Tfh cell fate. The regulatory network is complicated, and the number of novel molecules demonstrated to control the fate of Tfh cells is increasing. Therefore, this review aims to summarize the current knowledge regarding the molecular regulation of Tfh cell development and differentiation at the protein level and at the epigenetic level to elucidate Tfh cell biology and provide potential targets for clinical interventions in the future.

<b>Background:</b> Delaying umbilical cord clamping until after aeration of the lung (physiological-based cord clamping; PBCC) maintains cardiac output and oxygenation in preterm lambs at birth, however, its efficacy after intrauterine inflammation is not known. Given the high incidence of chorioamnionitis in preterm infants, we investigated whether PBCC conferred any benefits compared to immediate cord clamping (ICC) in preterm lambs exposed antenatally to 7 days of intrauterine inflammation. <b>Methods:</b> Ultrasound guided intraamniotic injection of 20 mg Lipopolysaccharide (from <i>E. coli</i>:055:B5) was administered to pregnant ewes at 0.8 gestation. Seven days later, ewes were anesthetized, preterm fetuses exteriorised via cesarean section, and instrumented for continuous measurement of pulmonary, systemic and cerebral pressures and flows, and systemic, and cerebral oxygenation. Lambs were then randomized to either PBCC, whereupon ventilation was initiated and maintained for 3 min prior to umbilical cord clamping, or ICC where the umbilical cord was cut and ventilation initiated 30 s later. Ventilation was maintained for 30 min. <b>Results:</b> ICC caused a rapid fall in systemic (by 25%) and cerebral (by 11%) oxygen saturation in ICC lambs, concurrent with a rapid increase in carotid arterial pressure and heart rate. The overshoot in carotid arterial pressure was sustained in ICC lambs for the first 20 min of the study. PBCC maintained cardiac output and prevented the fall in cerebral oxygen delivery at birth. PBCC lambs had lower respiratory compliance and higher respiratory requirements throughout the study. <b>Conclusion:</b> PBCC mitigated the adverse effects of ICC on oxygenation and cardiac output, and therefore could be more beneficial in preterm babies exposed to antenatal inflammation as it maintains cardiac output and oxygen delivery. The increased respiratory requirements require further investigation in this sub-group of preterm infants.

Extracorporeal membrane oxygenation (ECMO) is becoming increasingly utilized to manage neonates with cardiac and respiratory failure. The procedure involves extensive anticoagulation in a sick neonate with underlying disease pathology. In addition, the immature hemostatic system in the neonate adds to the complexity of titrating the necessary anticoagulation. This places the infant at greater risk for life threatening hemorrhage and thrombosis. Managing anticoagulation in these infants is extremely challenging and needs the expertise of a physician with a thorough knowledge of the intricacies of developmental hemostasis and limitations of the current laboratory techniques available to manage anticoagulation. This article provides a brief overview of the developing hemostatic system of the neonate and the challenges associated with managing anticoagulation in this vulnerable population of patients.

Generally repressed by epigenetic mechanisms, retrotransposons represent around 40% of the murine genome. At the Agouti viable yellow (A^vy) locus, an endogenous retrovirus (ERV) of the intracisternal A particle (IAP) class retrotransposed upstream of the agouti coat-color locus, providing an alternative promoter that is variably DNA methylated in genetically identical individuals. This results in variable expressivity of coat color that is inherited transgenerationally. Here, a systematic genome-wide screen identifies multiple C57BL/6J murine IAPs with A^vy epigenetic properties. Each exhibits a stable methylation state within an individual but varies between individuals. Only in rare instances do they act as promoters controlling adjacent gene expression. Their methylation state is locus-specific within an individual, and their flanking regions are enriched for CTCF. Variably methylated IAPs are reprogrammed after fertilization and re-established as variable loci in the next generation, indicating reconstruction of metastable epigenetic states and challenging the generalizability of non-genetic inheritance at these regions.

This book chapter describes the use of droplet microfluidics to phenotype single cells. The basic process flow includes the encapsulation of single cells with a specific probe into aqueous micro-droplets suspended in a biocompatible oil. The probe is chosen to measure the phenotype of interest. After incubation, the encapsulated cell turns the probe fluorescent and renders the entire droplet fluorescent. Enumerating drops that are fluorescent quantifies the concentration of cells possessing the phenotype of interest. Examining the distribution of fluorescence further allows one to quantify the heterogeneity among the cell population.

Introduction of polymeric nanoparticles in cancer therapeutics is widely investigated since nanomedicine often enables the intratumoral delivery of drugs with increased efficacy with minimal side effects. In this study MRI monitoring was employed to study the therapeutic effect of nanocombretastatin (G3-CA4) in an orthotopic glioma model. Water insoluble combretastatin (CA4) was conjugated to a small-sized water soluble G3-succinamic acid PAMAM dendrimer. Nanoconstruct sizes were determined by TEM to be 3 to 5 nm. Intravenous (i.v.) delivery of G3-CA4 in an orthotopic glioma model produced a long-lived ischemia accompanied by necrosis at the core of the tumor but leaving a rim of viable tissue. In contrast, delivery of CA4 alone has no therapeutic effect in an experimental rat model of glioma.

Recent experiments suggest that DNA-mediated charge transport might enable signaling between the [4Fe4S] clusters in the C-terminal domains of human DNA primase and polymerase α, as well as the signaling between other replication and repair high-potential [4Fe4S] proteins. Our theoretical study demonstrates that the redox signaling cannot be accomplished exclusively by DNA-mediated charge transport because part of the charge transfer chain has an unfavorable free energy profile. We show that hole or excess electron transfer between a [4Fe4S] cluster and a nucleic acid duplex through a protein medium can occur within microseconds in one direction, while it is kinetically hindered in the opposite direction. We present a set of signaling mechanisms that may occur with the assistance of oxidants or reductants, using the allowed charge transfer processes. These mechanisms would enable the coordinated action of [4Fe4S] proteins on DNA, engaging the [4Fe4S] oxidation state dependence of the protein-DNA binding affinity.

<h4>Background</h4>Many respiratory conditions have been attributed to toxic dust and fume exposure in World Trade Center (WTC) rescue and recovery workers, who frequently report symptoms of OSA. We examined the prevalence of new-onset OSA and tested if the prevalence and severity of OSA are related to the presence of chronic rhinosinusitis (CRS).<h4>Methods</h4>A total of 601 subjects (83% men; age, 33-87 years; BMI, 29.9 ± 5.5 kg/m²) enrolled in the WTC Health Program, excluding those with significant pre-September 11, 2001, snoring or prior CRS, underwent two nights of home sleep testing. OSA was defined as Apnea Hypopnea Index 4% ≥ 5 events/h or respiratory disturbance index of ≥ 15 events/h. CRS was assessed using nasal symptom questionnaires.<h4>Results</h4>The prevalence of OSA was 75% (25% no OSA, 46% mild OSA, 19% moderate OSA, and 10% severe OSA), and the prevalence of CRS was 43.5%. Compared with no CRS, new and worsening CRS was a significant risk factor for OSA with an OR of 1.80 (95% CI, 1.18-2.73; P = .006) unadjusted and 1.76 (95% CI, 1.08-2.88; P = .02) after adjustment for age, BMI, sex, gastroesophageal reflux disorder, and alcohol use.<h4>Conclusions</h4>The high prevalence of OSA in WTC responders was not explained fully by obesity and sex. Possible mechanisms for the elevated risk of OSA in subjects with CRS include increased upper airway inflammation and/or elevated nasal/upper airway resistance, but these need confirmation.

<h4>Objective</h4>This review was designed to identify highlights of the osteoarthritis (OA) genetics and epigenetics literature published between April 2017 and January 2018.<h4>Design</h4>A Pubmed literature search was conducted using the keywords 'osteoarthritis' and each of the following: 'genomic', 'genetic', 'epigenomic', 'epigenetic', 'histone', 'noncoding RNA', 'miRNA', 'lncRNA', 'DNA methylation', 'DNA hydroxymethylation', 'DNMT', and 'TET'. The dates of publication were restricted to 4/1/2017-1/15/2018. Results were compared to the same search terms limited to 4/1/2016-1/15/2017.<h4>Results</h4>Virtually all search term combinations demonstrated a decrease in papers published this year compared to last, with epigenetic and miRNA/lncRNA research being stable. Despite this, numerous advances were made this year, including the second large genome-wide association study (GWAS) study of hand OA, a new twin study of hip and knee OA concordance, an extensive study of GDF5 evolution, analyses of the contribution of Dnmt3a to OA, a description of DNA methylation in a nonhuman primate model of OA, and an integrated, multi-omics analysis of DNA methylation, mRNA, and protein expression in human OA samples, among others. A variety of micro- and a few circular-RNA studies were also published, highlighting the importance of noncoding RNA in both the pathogenesis and potential treatment of OA.<h4>Conclusion</h4>Although publications have decreased slightly in the last year, genetics and epigenetics continue to be a topic of substantial research in OA, and considerable progress continues to be made in the field.

No abstract available.

Due to the poor dispersion in polymer matrix, graphene can hardly be used alone as a flame-retardant additive for polymers. In this paper, a novel halogen-free flame retardant - the ternary graft product of silsesquioxane, graphene oxide and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (PMGO) with a structure of graphene oxide layers modified with the synergistic flame retardant multiple elements of phosphorous, nitrogen and silicon was synthesized and then used in combination with traditional intumescent flame retardant (IFR) to enhance the flame retardancy of polypropylene (PP). The experimental results show that the thermal and flame retardant properties of flame-retardant (FR) PP composites are significantly improved by introducing 5 wt% PMGO as well as 20 wt% IFR. The peak heat release rate and total heat release of the FR PP composite are reduced 61.5% and 40.2%, respectively, compared to neat PP. Based on the char layer observation and thermal analysis, the enhancement in flame retardancy is mainly attributed to the outstanding intumescent char layers with high strength and thermal stability formed under the synergistic effect of PMGO and IFR. Besides, the introduced phosphorous, nitrogen and silicon hydrophilic groups do not show the negative effects on the surface hydrophobicity of flame retardant PP materials, which could broaden its scope of application.

Huntington's disease (HD) is a monogenic, progressive, neurodegenerative disorder with currently no available treatment. The Libechov transgenic minipig model for HD (TgHD) displays neuroanatomical similarities to humans and exhibits slow disease progression, and is therefore more powerful than available mouse models for the development of therapy. The phenotypic characterization of this model is still ongoing, and it is essential to validate biomarkers to monitor disease progression and intervention. In this study, the behavioral phenotype (cognitive, motor and behavior) of the TgHD model was assessed, along with biomarkers for mitochondrial capacity, oxidative stress, DNA integrity and DNA repair at different ages (24, 36 and 48 months), and compared with age-matched controls. The TgHD minipigs showed progressive accumulation of the mutant huntingtin (mHTT) fragment in brain tissue and exhibited locomotor functional decline at 48 months. Interestingly, this neuropathology progressed without any significant age-dependent changes in any of the other biomarkers assessed. Rather, we observed genotype-specific effects on mitochondrial DNA (mtDNA) damage, mtDNA copy number, 8-oxoguanine DNA glycosylase activity and global level of the epigenetic marker 5-methylcytosine that we believe is indicative of a metabolic alteration that manifests in progressive neuropathology. Peripheral blood mononuclear cells (PBMCs) were relatively spared in the TgHD minipig, probably due to the lack of detectable mHTT. Our data demonstrate that neuropathology in the TgHD model has an age of onset of 48 months, and that oxidative damage and electron transport chain impairment represent later states of the disease that are not optimal for assessing interventions.This article has an associated First Person interview with the first author of the paper.

Passive immunotherapy, i.e., the administration of exogenous antibodies that recognize a specific target antigen, has gained significant momentum as a potential treatment strategy for several central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and brain cancer, among others. Advances in antibody engineering to create therapeutic antibody fragments or antibody conjugates have introduced new strategies that may also be applied to treat CNS disorders. However, drug delivery to the CNS for antibodies and other macromolecules has thus far proven challenging, due in large part to the blood-brain barrier and blood-cerebrospinal fluid barriers that greatly restrict transport of peripherally administered molecules from the systemic circulation into the CNS. Here, we summarize the various passive immunotherapy approaches under study for the treatment of CNS disorders, with a primary focus on disease-specific and target site-specific challenges to drug delivery and new, cutting edge methods.

No abstract available.

<h4>Background</h4>In animals with XY sex chromosomes, X-linked genes from a single X chromosome in males are imbalanced relative to autosomal genes. To minimize the impact of genic imbalance in male Drosophila, there is a dosage compensation complex (MSL) that equilibrates X-linked gene expression with the autosomes. There are other potential contributions to dosage compensation. Hemizygous autosomal genes located in repressive chromatin domains are often derepressed. If this homolog-dependent repression occurs on the X, which has no pairing partner, then derepression could contribute to male dosage compensation.<h4>Results</h4>We asked whether different chromatin states or topological associations correlate with X chromosome dosage compensation, especially in regions with little MSL occupancy. Our analyses demonstrated that male X chromosome genes that are located in repressive chromatin states are depleted of MSL occupancy; however, they show dosage compensation. The genes in these repressive regions were also less sensitive to knockdown of MSL components.<h4>Conclusions</h4>Our results suggest that this non-canonical dosage compensation is due to the same transacting derepression that occurs on autosomes. This mechanism would facilitate immediate compensation during the evolution of sex chromosomes from autosomes. This mechanism is similar to that of C. elegans, where enhanced recruitment of X chromosomes to the nuclear lamina dampens X chromosome expression as part of the dosage compensation response in XX individuals.

High-performance bioceramics are required for preventing failure and prolonging the life-time of bone grafting scaffolds and osseous implants. The proper identification and development of materials with extended functionalities addressing socio-economic needs and health problems constitute important and critical steps at the heart of clinical research. Recent findings in the realm of ion-substituted hydroxyapatite (HA) could pave the road towards significant developments in biomedicine, with an emphasis on a new generation of orthopaedic and dentistry applications, since such bioceramics are able to mimic the structural, compositional and mechanical properties of the bone mineral phase. In fact, the fascinating ability of the HA crystalline lattice to allow for the substitution of calcium ions with a plethora of cationic species has been widely explored in the recent period, with consequent modifications of its physical and chemical features, as well as its functional mechanical and in vitro and in vivo biological performance. A comprehensive inventory of the progresses achieved so far is both opportune and of paramount importance, in order to not only gather and summarize information, but to also allow fellow researchers to compare with ease and filter the best solutions for the cation substitution of HA-based materials and enable the development of multi-functional biomedical designs. The review surveys preparation and synthesis methods, pinpoints all the explored cation dopants, and discloses the full application range of substituted HA. Special attention is dedicated to the antimicrobial efficiency spectrum and cytotoxic trade-off concentration values for various cell lines, highlighting new prophylactic routes for the prevention of implant failure. Importantly, the current in vitro biological tests (widely employed to unveil the biological performance of HA-based materials), and their ability to mimic the in vivo biological interactions, are also critically assessed. Future perspectives are discussed, and a series of recommendations are underlined.

The role of genetic variability in dementia with Lewy bodies (DLB) is now indisputable; however, data regarding copy number variation (CNV) in this disease has been lacking. Here, we used whole-genome genotyping of 1454 DLB cases and 1525 controls to assess copy number variability. We used 2 algorithms to confidently detect CNVs, performed a case-control association analysis, screened for candidate CNVs previously associated with DLB-related diseases, and performed a candidate gene approach to fully explore the data. We identified 5 CNV regions with a significant genome-wide association to DLB; 2 of these were only present in cases and absent from publicly available databases: one of the regions overlapped LAPTM4B, a known lysosomal protein, whereas the other overlapped the NME1 locus and SPAG9. We also identified DLB cases presenting rare CNVs in genes previously associated with DLB or related neurodegenerative diseases, such as SNCA, APP, and MAPT. To our knowledge, this is the first study reporting genome-wide CNVs in a large DLB cohort. These results provide preliminary evidence for the contribution of CNVs in DLB risk.

This data article provides raw auditory evoked brainstem responses (ABRs) from controls and Ca_v2.3 transgenics, i.e. heterozygous Ca_v2.3^+/- and Ca_v2.3^-/- null mutants. Gender specific ABR recordings were performed in age-matched animals under ketamine/xylazine narcosis. Data presented here include ABRs upon both click and tone burst presentation in the increasing SPL mode using a commercially available ABR setup from Tucker Davis Technologies Inc. (TDT, USA). Detailed information is provided for the sound attenuating cubicle, electrical shielding, electrode parameters, stimulus characteristics and architecture, sampling rate, filtering processes and ABR protocol application during the course of data acquisition and recording. The later are important for subsequent analysis of click and tone burst related hearing thresholds, amplitude growth function and peak latencies. Raw data are available at MENDELEY DATA, DIO: 〈DOI:10.17632/g6ygz2spzx.1〉, URL: 〈https://data.mendeley.com/datasets/g6ygz2spzx/1〉).

Human milk is uniquely optimized for the needs of the developing infant. Its composition is complex and dynamic, driven primarily by maternal genetics, and to a lesser extent by diet and environment. One important component that is gaining attention is the milk fat globule (MFG). The MFG is composed of a triglyceride-rich core surrounded by a tri-layer membrane, also known as the milk fat globule membrane (MFGM) that originates from mammary gland epithelia. The MFGM is enriched with glycerophospholipids, sphingolipids, cholesterol, and proteins, some of which are glycosylated, and are known to exert numerous biological roles. Mounting evidence suggests that the structure of the MFG and bioactive components of the MFGM may benefit the infant by aiding in the structural and functional maturation of the gut through the provision of essential nutrients and/or regulating various cellular events during infant growth and immune education. Further, antimicrobial peptides and surface carbohydrate moieties surrounding the MFG might have a pivotal role in shaping gut microbial populations, which in turn may promote protection against immune and inflammatory diseases early in life. This review seeks to: (1) understand the components of the MFG, as well as maternal factors including genetic and lifestyle factors that influence its characteristics; (2) examine the potential role of this milk component on the intestinal immune system; and (3) delineate the mechanistic roles of the MFG in infant intestinal maturation and establishment of the microbiota in the alimentary canal.

<h4>Background</h4>The current classification of human lung adenocarcinoma defines five different histological growth patterns within the group of conventional invasive adenocarcinomas. The five growth patterns are characterised by their typical architecture, but also by variable tumor biological behaviour.<h4>Aims</h4>The aim of this study was to identify specific gene signatures of the five adenocarcinoma growth patterns defined by the joint IASLC/ATS/ERS working group.<h4>Methods</h4>Total RNA from microdissected adenocarcinoma tissue samples of ten lepidic, ten acinar, ten solid, nine papillary, and nine micropapillary tumor portions was isolated and prepared for gene expression analysis. Differential expression of genes was determined using the R package "LIMMA". The overall significance of each signature was assessed via global test. Gene ontology statistics were analysed using GOstat. For immunohistochemical validation, tissue specimens from 20 tumors with solid and 20 tumors with lepidic growth pattern were used.<h4>Results</h4>Microarray analyses between the growth patterns resulted in numerous differentially expressed genes between the solid architecture and other patterns. The comparison of transcriptomic activity in the solid and lepidic patterns revealed 705 up- and 110 downregulated non-redundant genes. The pattern-specific protein expression of Inositol-1,4,5-trisphosphate-kinase-A (ITPKA) and angiogenin by immunohistochemistry confirmed the RNA levels. The strongest differences in protein expression between the two patterns were shown for ITPKA (p = 0.02) and angiogenin (p = 0.113).<h4>Conclusions</h4>In this study growth pattern-specific gene signatures in pulmonary adenocarcinoma were identified and distinct transcriptomic differences between lung adenocarcinoma growth patterns were defined. The study provides valuable new information about pulmonary adenocarcinoma and allows a better assessment of the five adenocarcinoma subgroups.

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

<h4>Background</h4>Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL, OMIM #611105) is a genetic disease of the central nervous system characterized by lower limb spasticity, cerebellar ataxia and involvement of the dorsal column. The disease is caused by mutations in the DARS2 gene but has never been reported in sub-Saharan Africa so far.<h4>Case presentation</h4>Two siblings, aged 18 years and 15 years, from a consanguineous family presented with pyramidal signs and symptoms since infancy and developmental delay. Whole exome sequencing of the proband identified two compound heterozygous variants (NM_018122.4:c.1762C > G and c.563G > A) in DARS2. Sanger sequencing confirmed the presence of the mutations and their segregation in trans in both patients and in their elder sister (aged 20 years), who showed only brisk reflexes and mild lower limb spasticity. Surprisingly, in contrast to her subtle clinical presentation, the elder sister had abnormal MRI features and serum lactate levels comparable to her ill sisters.<h4>Conclusion</h4>This report illustrates intra-familial phenotypic variation in LBSL and provides an example of a marked dissociation between the clinical and radiological phenotypes of the disease. This may have implications for the detection of mutation carriers in LBSL.

Short tandem repeats (STRs) are involved in dozens of Mendelian disorders and have been implicated in complex traits. However, genotyping arrays used in genome-wide association studies focus on single nucleotide polymorphisms (SNPs) and do not readily allow identification of STR associations. We leverage next-generation sequencing (NGS) from 479 families to create a SNP + STR reference haplotype panel. Our panel enables imputing STR genotypes into SNP array data when NGS is not available for directly genotyping STRs. Imputed genotypes achieve mean concordance of 97% with observed genotypes in an external dataset compared to 71% expected under a naive model. Performance varies widely across STRs, with near perfect concordance at bi-allelic STRs vs. 70% at highly polymorphic repeats. Imputation increases power over individual SNPs to detect STR associations with gene expression. Imputing STRs into existing SNP datasets will enable the first large-scale STR association studies across a range of complex traits.

Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.

<h4>Objective</h4>To compare neurodevelopmental outcomes in linear growth-restricted (LGR) infants born <29 weeks with and without weight gain out of proportion to linear growth.<h4>Study design</h4>We compared 2-year neurodevelopmental outcomes between infants with and without LGR and between LGR infants with and without weight gain out of proportion to linear growth. The outcomes were Bayley-III cognitive, motor, and language scores, cerebral palsy, Gross Motor Function Classification System (GMFCS) level ≥ 2, and neurodevelopmental impairment.<h4>Result</h4>In total, 1227 infants were analyzed. LGR infants were smaller and less mature at birth, had higher BMI, and had lower Bayley-III language scores (82.3 vs. 85.0, p < 0.05). Among infants with LGR, infants with high BMI had lower language scores compared with those with low-to-normal BMI (80.8 vs. 83.3, p < 0.05), and were more likely to have GMFCS level ≥2 and neurodevelopmental impairment.<h4>Conclusion</h4>Among infants with LGR, weight gain out of proportion to linear growth was associated with poorer neurodevelopmental outcomes.

<h4>Abstarct</h4>OBJECTIVE: The diagnosis of neonatal-onset protein C (PC) deficiency is challenging. This study aimed to establish the neonatal screening of heritable PC deficiency in Japan.<h4>Study design</h4>We determined the changes in plasma activity levels of PC and protein S (PS) in healthy neonates, and studied newborn patients with PROC mutation in the Japanese registry.<h4>Result</h4>Physiological PC and PS levels increased with wide range. The PC/PS-activity ratios converged after birth. The PC/PS-activity ratios of 19 patients with biallelic mutations, but not, 9 with monoallelic mutation, were lower than those of 13 without mutation. The logistic regression analyses established a formula including two significant variables of PC activity (cut-off < 10%, odds ratio = 30.0) and PC/PS-activity ratio (cut-off < 0.35, odds ratio = 22.7), with 93% sensitivity and 44% specificity for determining patients with mutation(s).<h4>Conclusion</h4>The PC/PS-activity ratio is an effective parameter for the genetic screening of neonatal-onset PC-deficiency in Japanese population.

The lack of consensus on bone marrow (BM) and splenic immune cell profiles in preclinical mouse strains complicates comparative analysis across different studies. Although studies have documented relative distribution of immune cells from peripheral blood in mice, similar studies for BM and spleen from naïve mice are lacking. In an effort to establish strain- and gender-specific benchmarks for distribution of various immune cell subtypes in these organs, we performed immunophenotypic analysis of BM cells and splenocytes from both genders of three commonly used murine strains (C57BL/6NCr, 129/SvHsd, and BALB/cAnNCr). Total neutrophils and splenic macrophages were significantly higher in C57BL/6NCr, whereas total B cells were lower. Within C57BL/6NCr female mice, BM B cells were elevated with respect to the males whereas splenic mDCs and splenic neutrophils were reduced. Within BALB/cAnNCr male mice, BM CD4⁺ Tregs were elevated with respect to the other strains. Furthermore, in male BALB/cAnNCr mice, NK cells were elevated with respect to the other strains in both BM and spleen. Splenic CD4⁺ Tregs and splenic CD8⁺ T cells were reduced in male BALB/c mice in comparison to female mice. Bone marrow CD4⁺ T cells and mDCs were significantly increased in 129/SvHsd whereas splenic CD8⁺ T cells were reduced. In general, males exhibited higher immature myeloid cells, macrophages, and NK cells. To our knowledge, this study provides a first attempt to systematically establish organ-specific benchmarks on immune cells in studies involving these mouse strains.

Nuclear receptor coactivator 4 (NCOA4) is a selective cargo receptor that mediates the autophagic degradation of ferritin ("ferritinophagy"), the cytosolic iron storage complex. NCOA4-mediated ferritinophagy maintains intracellular iron homeostasis by facilitating ferritin iron storage or release according to demand. Ferritinophagy is involved in iron-dependent physiological processes such as erythropoiesis, where NCOA4 mediates ferritin iron release for mitochondrial heme synthesis. Recently, ferritinophagy has been shown to regulate ferroptosis, a newly described form of iron-dependent cell death mediated by excess lipid peroxidation. Dysregulation of iron metabolism and ferroptosis have been described in neurodegeneration, cancer, and infection, but little is known about the role of ferritinophagy in the pathogenesis of these diseases. Here, we will review the biochemical regulation of NCOA4, its contribution to physiological processes and its role in disease. Finally, we will discuss the potential of activating or inhibiting ferritinophagy and ferroptosis for therapeutic purposes.

Iron homeostasis is a tightly regulated process in all living organisms because this metal is essential for cellular metabolism, but could be extremely toxic when present in excess. In mammals, there is a complex pathway devoted to iron regulation, whose key protein is hepcidin (Hepc), which is a powerful iron absorption inhibitor mainly produced by the liver. Transferrin receptor 2 (Tfr2) is one of the hepcidin regulators, and mutations in <i>TFR2</i> gene are responsible for type 3 hereditary hemochromatosis (HFE3), a genetically heterogeneous disease characterized by systemic iron overload. It has been recently pointed out that Hepc production and iron regulation could be exerted also in tissues other than liver, and that Tfr2 has an extrahepatic role in iron metabolism as well. This review summarizes all the most recent data on Tfr2 extrahepatic role, taking into account the putative distinct roles of the two main Tfr2 isoforms, Tfr2α and Tfr2β. Representing Hepc modulation an effective approach to correct iron balance impairment in common human diseases, and with Tfr2 being one of its regulators, it would be worthwhile to envisage Tfr2 as a therapeutic target.

During the last decade, autophagy has been pointed out as a central process in cellular homeostasis with the consequent implication in most cellular settings and human diseases pathology. At present, there is significant data available about molecular mechanisms that regulate autophagy. Nevertheless, autophagy pathway itself and its importance in different cellular aspects are still not completely clear. In this article, we are focused in four main aspects: (a) <i>Induction of Autophagy</i>: Autophagy is an evolutionarily conserved mechanism induced by nutrient starvation or lack of growth factors. In higher eukaryotes, autophagy is a cell response to stress which starts as a consequence of organelle damage, such as oxidative species and other stress conditions. (b) <i>Initiation of Autophagy</i>; The two major actors in this signaling process are mTOR and AMPK. These multitasking protein complexes are capable to summarize the whole environmental, nutritional, and energetic status of the cell and promote the autophagy induction by means of the ULK1-Complex, that is the first member in the autophagy initiation. (c) <i>ULK1-Complex:</i> This is a highly regulated complex responsible for the initiation of autophagosome formation. We review the post-transductional modifications of this complex, considering the targets of ULK1. (d)<i>The mechanisms involved in autophagosome formation</i>. In this section we discuss the main events that lead to the initial structures in autophagy. The BECN1-Complex with PI3K activity and the proper recognition of PI3P are one of these. Also, the transmembrane proteins, such as VMP1 and ATG9, are critically involved. The membrane origin and the cellular localization of autophagosome biogenesis will be also considered. Hence, in this article we present an overview of the current knowledge of the molecular mechanisms involved in the initial steps of mammalian cell autophagosome biogenesis.

Polymersomes are attractive nanocarriers for hydrophilic and lipophilic drugs; they are more stable than liposomes, tunable, and relatively easy to prepare. The copolymer composition and molar mass are critical features that determine the physicochemical properties of the polymersomes including the rate of drug release. We used the triblock-copolymer, poly(2-methyl-2-oxazoline)-<i>block</i>-poly-(dimethysiloxane)-<i>block</i>-poly(2-methyl-2-oxazoline) (PMOXA-PDMS-PMOXA), to form amphipathic polymersomes capable of loading proteins and small hydrophobic agents. The selected agents were unstable neurotrophins (nerve growth factor and brain-derived neurotrophic factor), a large protein CD109, and the fluorescent drug curcumin. We prepared, characterized, and tested polymersomes loaded with selected agents in 2D and 3D biological models. Curcumin-loaded and rhodamine-bound PMOXA-PDMS-PMOXA polymersomes were used to visualize them inside cells. <i>N</i>-Methyl-d-aspartate receptor (NMDAR) agonists and antagonists were also covalently attached to the surface of polymersomes for targeting neurons. Labeled and unlabeled polymersomes with or without loaded agents were characterized using dynamic light scattering (DLS), UV-vis fluorescence spectroscopy, and asymmetrical flow field-flow fractionation (AF₄). Polymersomes were imaged and tested for biological activity in human and murine fibroblasts, murine macrophages, primary murine dorsal root ganglia, and murine hippocampal cultures. Polymersomes were rapidly internalized and there was a clear intracellular co-localization of the fluorescent drug (curcumin) with the fluorescent rhodamine-labeled polymersomes. Polymersomes containing CD109, a glycosylphosphatidylinositol-anchored protein, promoted cell migration in the model of wound healing. Nerve growth factor-loaded polymersomes effectively enhanced neurite outgrowth in dissociated and explanted dorsal root ganglia. Brain-derived neurotrophic factor increased dendritic spine density in serum-deprived hippocampal slice cultures. NMDAR agonist- and antagonist-functionalized polymersomes targeted selectively neurons over glial cells in mixed cultures. Collectively, the study reveals the successful incorporation into polymersomes of biologically active trophic factors and small hydrophilic agents that retain their biological activity in vitro, as demonstrated in selected central and peripheral tissue models.

<h4>Background and aims</h4>Depression and diabetes are becoming increasingly prevalent within the Vietnamese elderly population. However, the linkage between these health conditions in the Vietnamese elderly has not yet been fully investigated. This study aimed to assess the level of depressive symptoms and associated factors among elderly diabetic patients.<h4>Methods</h4>A cross-sectional study was conducted at National Geriatric Hospital in the elder patients aged ≥60 years with type 2 diabetes mellitus (T2DM). Depressive symptoms were assessed using the Geriatric Depression Scale, with three categories: normal (0-4 points), mild (5-9 points), and moderate/severe depressive symptoms (≥10 points). We obtained information on the patient's sociodemographic, medical history, glycemic control (fasting plasma glucose and HbA_1c), daily activities (activities of daily living [ADLs] and instruments activities of daily living [IADLs] scale), and fall risks (Time Up and Go test). Logistic regression was used to analyze the factors associated with the presence of depressive symptoms.<h4>Results</h4>Among 412 patients, 236 (57.3%) had HbA_1c level at 7.0% or higher. There were 327 (79.4%) patients having depressive symptoms. The level of HbA_1c was significantly different between the depressive symptom group and the non-depressive symptom group (7.74% and 6.61%, <i>P</i><0.05). The increased likelihood of having depressive symptoms was associated with having risk of falls (OR: 5.50; 95% CI: 1.88-16.11), suffering from 5-10 years of diabetes (OR: 2.74, 95% CI: 1.28-5.85), uncontrolled fasting plasma glucose (OR: 4.06, 95% CI: 1.81-9.12), and an impairment of IADLs (OR: 5.74, 95% CI: 2.24-14.7).<h4>Conclusion</h4>This study highlights a high prevalence of depressive symptoms among elderly T2DM patients in Vietnam, suggesting an urgent need for screening depressive symptoms and providing mental health care services to this population promptly, particularly to those suffering from diabetes for a long period of time or co-functional impairments.

Dual action agents containing a cysteine protease inhibitor and Ru-based photosensitizer for photodynamic therapy (PDT) were designed, synthesized, and validated in 2D culture and 3D functional imaging assays of triple-negative human breast cancer (TNBC). These combination agents deliver and release Ru-based PDT agents to tumor cells and cause cancer cell death upon irradiation with visible light, while at the same time inactivating cathespin B (CTSB), a cysteine protease strongly associated with invasive and metastatic behavior. In total five Ru-based complexes were synthesized with the formula [Ru(bpy)₂(1)](O₂CCF₃)₂ (3), where bpy = 2,2'-bipyridine and 1 = a bipyridine-based epoxysuccinyl inhibitor; [Ru(tpy)(NN)(2)](PF₆)₂, where tpy = terpiridine, 2 = a pyridine-based epoxysuccinyl inhibitor and NN = 2,2'-bipyridine (4); 6,6'-dimethyl-2,2'-bipyridine (5); benzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (6); and 3,6-dimethylbenzo[ i]dipyrido[3,2- a:2',3'- c]phenazine (7). Compound 3 contains a [Ru(bpy)₃]²⁺ fluorophore and was designed to track the subcellular localization of the conjugates, whereas compounds 4-7 were designed to undergo either photoactivated ligand dissociation and/or singlet oxygen generation. Photochemical studies confirmed that complexes 5 and 7 undergo photoactivated ligand dissociation, whereas 6 and 7 generate singlet oxygen. Inhibitors 1-7 all potently and irreversibly inhibit CTSB. Compounds 4-7 were evaluated against MDA-MB-231 TNBC and MCF-10A breast epithelial cells in 2D and 3D culture for effects on proteolysis and cell viability under dark and light conditions. Collectively, these data reveal that 4-7 potently inhibit dye-quenched (DQ) collagen degradation, whereas only compound 7 causes efficient cell death under light conditions, consistent with its ability to release a Ru(II)-based photosensitizer and to also generate ¹O₂.

<h4>Background</h4>Non-alcoholic fatty liver disease (NAFLD) encompasses benign steatosis and more severe conditions such as non-alcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. This chronic liver disease has a poorly understood etiology and demonstrates sexual dimorphisms. We aim to examine the molecular mechanisms underlying sexual dimorphisms in NAFLD pathogenesis through a comprehensive multi-omics study. We integrated genomics (DNA variations), transcriptomics of liver and adipose tissue, and phenotypic data of NAFLD derived from female mice of ~ 100 strains included in the hybrid mouse diversity panel (HMDP) and compared the NAFLD molecular pathways and gene networks between sexes.<h4>Results</h4>We identified both shared and sex-specific biological processes for NAFLD. Adaptive immunity, branched chain amino acid metabolism, oxidative phosphorylation, and cell cycle/apoptosis were shared between sexes. Among the sex-specific pathways were vitamins and cofactors metabolism and ion channel transport for females, and phospholipid, lysophospholipid, and phosphatidylinositol metabolism and insulin signaling for males. Additionally, numerous lipid and insulin-related pathways and inflammatory processes in the adipose and liver tissue appeared to show more prominent association with NAFLD in male HMDP. Using data-driven network modeling, we identified plausible sex-specific and tissue-specific regulatory genes as well as those that are shared between sexes. These key regulators orchestrate the NAFLD pathways in a sex- and tissue-specific manner. Gonadectomy experiments support that sex hormones may partially underlie the sexually dimorphic genes and pathways involved in NAFLD.<h4>Conclusions</h4>Our multi-omics integrative study reveals sex- and tissue-specific genes, processes, and networks underlying sexual dimorphism in NAFLD and may facilitate sex-specific precision medicine.

Condensins play a unique role in orchestrating the global folding of the chromosome, an essential cellular process, and contribute to human disease and bacterial pathogenicity. As such, they represent an attractive and as yet untapped target for diverse therapeutic interventions. We describe here the discovery of small molecule inhibitors of the Escherichia coli condensin MukBEF. Pilot screening of a small diversity set revealed five compounds that inhibit the MukBEF pathway, two of which, Michellamine B and NSC260594, affected MukB directly. Computer-assisted docking suggested plausible binding sites for the two compounds in the hinge and head domains of MukB, and both binding sites were experimentally validated using mutational analysis and inspection of NSC260594 analogs. These results outline a strategy for the discovery of condensin inhibitors, identify druggable binding sites on the protein, and describe two small molecule inhibitors of condensins.

(+)-Hongoquercin A and B were synthesized from commercially available trans, trans-farnesol in six and eleven steps, respectively, using dual biomimetic strategies with polyketide aromatization and subsequent polyene functionalization from a common farnesyl-resorcylate intermediate. Key steps involve Pd(0)-catalyzed decarboxylative allylic rearrangement of a dioxinone β,δ-diketo ester to a β,δ-diketo dioxinone, which was readily aromatized into the corresponding resorcylate, and subsequent polyene cyclization via enantioselective protonation or regioselective terminal alkene oxidation and cationic cyclization of enantiomerically enriched epoxide to furnish the tetracyclic natural product cores. Analogues of the hongoquercin were synthesized via halonium-induced polyene cyclizations, and the meroterpenoid could be further functionalized via saponification, hydrolytic decarboxylation, reduction, and amidation reactions.

Copper-containing enzymes perform fundamental functions by activating dioxygen (O₂) and therefore allowing chemical energy-transfer for aerobic metabolism. The copper-dependence of O₂ transport, metabolism and production of signalling molecules are supported by molecular systems that regulate and preserve tightly-bound static and weakly-bound dynamic cellular copper pools. Disruption of the reducing intracellular environment, characterized by glutathione shortage and ambient Cu(II) abundance drives oxidative stress and interferes with the bidirectional, copper-dependent communication between neurons and astrocytes, eventually leading to various brain disease forms. A deeper understanding of of the regulatory effects of copper on neuro-glia coupling via polyamine metabolism may reveal novel copper signalling functions and new directions for therapeutic intervention in brain disorders associated with aberrant copper metabolism.

Although many cases of genetic adaptations to high elevations have been reported, the processes driving these modifications and the pace of their evolution remain unclear. Many high-elevation adaptations (HEAs) are thought to have arisen in situ as populations rose with growing mountains. In contrast, most high-elevation lineages of the Qinghai-Tibetan Plateau appear to have colonized from low-elevation areas. These lineages provide an opportunity for studying recent HEAs and comparing them with ancestral low-elevation alternatives. Herein, we compare four frogs (three species of <i>Nanorana</i> and a close lowland relative) and four lizards (<i>Phrynocephalus</i>) that inhabit a range of elevations on or along the slopes of the Qinghai-Tibetan Plateau. The sequential cladogenesis of these species across an elevational gradient allows us to examine the gradual accumulation of HEA at increasing elevations. Many adaptations to high elevations appear to arise gradually and evolve continuously with increasing elevational distributions. Numerous related functions, especially DNA repair and energy metabolism pathways, exhibit rapid change and continuous positive selection with increasing elevations. Although the two studied genera are distantly related, they exhibit numerous convergent evolutionary changes, especially at the functional level. This functional convergence appears to be more extensive than convergence at the individual gene level, although we found 32 homologous genes undergoing positive selection for change in both high-elevation groups. We argue that species groups distributed along a broad elevational gradient provide a more powerful system for testing adaptations to high-elevation environments compared with studies that compare only pairs of high-elevation versus low-elevation species.

Neurodegenerative disorders, such as Huntington's diseases and spinocerebellar ataxias (SCAs), are driven by proteins with expanded polyglutamine (polyQ) tracts. Recently, coiled-coil structures in polyQ regions of such proteins were shown to facilitate aggregate formation and ultimately lead to cell death. However, the molecular mechanism linking these structural domains to neuronal toxicity of polyQ proteins remains elusive. Here, we demonstrate that coiled-coil structures in the Q repeat region of SCA type 3 (SCA3) polyQ proteins confer protein toxicity in <i>Drosophila</i> neurons. To functionally characterize coiled-coil structures in the Q repeat regions, we generated three structural variants of SCA3 polyQ proteins: (<i>i</i>) MJDtr-76Q, containing both α-helical coiled-coil and β-sheet hairpin structures in the Q repeat region; (<i>ii</i>) MJDtr-70Q_cc0, possessing only α-helical coiled-coil structures due to the incorporation of β-sheet-breaking residues (Q-to-N or Q-to-E mutations); and (<i>iii</i>) MJDtr-70Q_pQp, with no secondary structure due to the introduced proline residues (Q-to-P mutations). Through comparative analysis of these variants, we found that coiled-coil structures facilitated nuclear localization of SCA3 polyQ proteins and induced dendrite defects in <i>Drosophila</i> dendritic arborization neurons. Furthermore, genetic and functional screening identified the transcription factor Foxo as a target of polyQ proteins, and coiled-coil-mediated interactions of Foxo and polyQ proteins in the nucleus resulted in the observed dendrite and behavioral defects in <i>Drosophila</i> These results demonstrate that coiled-coil structures of polyQ proteins are crucial for their neuronal toxicity, which is conferred through coiled-coil to coiled-coil interactions with the nuclear targets of these proteins.

During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in <i>Caenorhabditis elegans</i>, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in <i>C. elegans</i>.

The Hippo pathway is involved in intestinal epithelial homeostasis with Wnt, BMP, Notch, and EGF signaling. We investigated the relationship between Hippo and other signaling pathways and the role of MOB kinase activator 1A/1B (MOB1A/B) in intestinal homeostasis. Mice with intestinal epithelial cell (IEC)-specific depletion of MOB1A/B showed hyperproliferation in IECs, defects in secretory lineage differentiation and loss of intestinal stem cells and eventually died at 10-12 days after tamoxifen treatment. In MOB1A/B-depleted IECs, expression of Wnt target genes were downregulated but Bmp2 and Tgfbr2 were transcriptionally activated with enhanced YAP activity. In in vivo and in vitro experiments with several signaling inhibitors, it has been shown that the BMP inhibitor LDN193189 or TGF-β inhibitor SB431542 had effects on partial restoration of the intestinal degenerative phenotype. Treatment with these inhibitors restored differentiation of secretory lineage cells in MOB1A/B-deficient mice, but not ISC pools in the crypt region. These studies reveal that IEC-specific depletion of MOB1A/B induced overexpression of Bmp2 and Tgfbr2 and inhibited Wnt activity, finally leading to loss of ISCs and functional epithelia in the mouse intestine. These results suggest that MOB1A/B has an essential function for intestinal epithelial homeostasis by regulating YAP, Wnt activity, and BMP/TGF-β signaling.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.

Macaca fascicularis is a highly advantageous model in which to study human cochlea with regard to both evolutionary proximity and physiological similarity of the auditory system. To better understand the properties of primate cochlear function, we analyzed the genes predominantly expressed in M. fascicularis cochlea. We compared the cochlear transcripts obtained from an adult male M. fascicularis by macaque and human GeneChip microarrays with those in multiple macaque and human tissues or cells and identified 344 genes with expression levels more than 2-fold greater than in the other tissues. These "cochlear signature genes" included 35 genes responsible for syndromic or nonsyndromic hereditary hearing loss. Gene set enrichment analysis revealed groups of genes categorized as "ear development" and "ear morphogenesis" in the top 20 gene ontology categories in the macaque and human arrays, respectively. This dataset will facilitate both the study of genes that contribute to primate cochlear function and provide insight to discover novel genes associated with hereditary hearing loss that have yet to be established using animal models.

Current cure rates for retinoblastoma (RB) are very high in developed countries. Nonetheless, in less privileged places worldwide, delayed diagnosis and refusal to adhere to treatment still endure an obstacle to improve overall patient survival. Thus, the access to consistent biomarkers for diagnosis at an earlier stage may facilitate treatment and improve outcomes. Over recent years, much attention has been focused on miRNAs, key post-transcriptional regulators that when altered, largely contribute to carcinogenesis and tumor progression. Many of the ~ 2500 microRNAs described in humans have shown differential expression profiles in tumors. In this review, we summarize current data about the roles of miRNAs in RB along with their value as diagnostic/prognostic factors using electronic databases such as PubMed. We reviewed the importance of miRNA in RB biology and discussed their implications in clinic intervention. Several miRNAs have pointed out reliable diagnostic and prognostic molecular biomarkers. The emergence of targeted therapies has significantly improved cancer treatment. In the near future, the modulation of miRNAs will represent a good treatment strategy.

<h4>Background</h4>Current staging of pathological stage III colon cancer (CC) is suboptimal; many patients recur despite unremarkable preoperative staging. We previously reported that early postoperative PET-CT can alter the stage and management of up to 15% of patients with high-risk stage III CC. This study aimed to determine the role of the test in the general stage III CC population.<h4>Methods</h4>A retrospective study of all consecutive patients with stage III CC who underwent early postoperative PET-CT between 2005 and 2017.<h4>Results</h4>A total of 342 patients, 166 (48.5%) males, median age 66 years (range, 29-90), were included. Pathological stage was IIIA, IIIB, and IIIC in 18 (5.3%), 257 (75.1%), and 67 (19.6%) patients, respectively. Median number of positive lymph nodes was 2 (range, 0-32). PET-CT results modified the management of 46 patients (13.4%): 37 (10.8%) with overt metastatic disease and 9 (2.6%) with a second primary. The 5-year disease-free survival for true stage III patients was 81%. The median overall survival for the entire cohort and for true stage III patients was not reached and was 57.2 months for true stage IV. Of the 37 patients found to be metastatic, 14 (37.8%) underwent curative treatments and 9/14 (64.3%) remain disease-free, with a median follow-up of 83.8 months. Predictive factors for upstaging following PET-CT were identified.<h4>Conclusion</h4>Early postoperative PET-CT changed the staging and treatment of 13.4% of stage III CC patients and has the potential for early detection of curable metastatic disease. Outcome results are encouraging. Prospective validation is ongoing.

Iron is essential for life, while excess iron can be toxic. Iron generates hydroxyl radical, which is the most reactive free radical, causing oxidative stress. Since iron is absorbed through the diet but not excreted from the body, it accumulates with age in tissues, including the retina, consequently leading to age-related toxicity. This accumulation is further promoted by inflammation. Hereditary diseases such as aceruloplasminemia, Friedreich's ataxia, pantothenate kinase-associated neurodegeneration, and posterior column ataxia with retinitis pigmentosa involve retinal degeneration associated with iron dysregulation. In addition to hereditary causes, dietary or parenteral iron supplementation has been recently reported to elevate iron levels in the retinal pigment epithelium (RPE) and promote retinal degeneration. Ocular siderosis from intraocular foreign bodies or subretinal hemorrhage can also lead to retinopathy. Evidence from mice and humans suggests that iron toxicity may contribute to age-related macular degeneration pathogenesis. Iron chelators can protect photoreceptors and RPE in various mouse models. The therapeutic potential for iron chelators is under investigation.

Juvenile hemochromatosis is a rare autosomal recessive disease due to variants in the Hemojuvelin (HJV) gene. Although biological features mimic HFE hemochromatosis, clinical presentation is worst with massive iron overload diagnosed during childhood. Our study describes clinical features and results of genetic testing for a group of patients initially referred for a hepcidino-deficiency syndrome and for whom HJV hemochromatosis was finally diagnosed. 662 patients with iron overload and high serum transferrin saturation were tested, and five genes (HFE, HJV, HAMP, TFR2, SLC40A1) were sequenced. Among our cohort, ten unrelated patients were diagnosed with HJV hemochromatosis. Genetic testing revealed five previously published and five undescribed variants: p.Arg41Pro, p.His180Arg, p.Lys299Glu, p.Cys361Arg and p.Ala384Val. Surprisingly, this study revealed a late age of onset in some patients, contrasting with the commonly accepted definition of "juvenile" hemochromatosis. Five of our patients were 30 years old or older, including two very late discoveries. Biological features and severity of iron overload were similar in younger and older patients. Our study brings new insight on HJV hemochromatosis showing that mild phenotype and late onset are possible. Genetic testing for HJV variants should thus be performed for all patients displaying a non-p.Cys282Tyr homozygous HFE hemochromatosis with hepcidin deficiency phenotype.

Resveratrol (RSV) is a small compound first identified as an activator of sirtuin 1 (SIRT1), a key factor in mediating the effects of caloric restriction. Since then, RSV received great attention for its widespread beneficial effects on health and in connection to many diseases. RSV improves the metabolism and the mitochondrial function, and more recently it was shown to restore fatty acid β-oxidation (FAO) capacities in patient fibroblasts harboring mutations with residual enzyme activity. Many of RSV's beneficial effects are mediated by the transcriptional coactivator PGC-1α, a direct target of SIRT1 and a master regulator of the mitochondrial fatty acid oxidation. Despite numerous studies RSV's mechanism of action is still not completely elucidated. Our aim was to investigate the effects of RSV on gene regulation on a wide scale, possibly to detect novel genes whose up-regulation by RSV may be of interest with respect to disease treatment. We performed Next Generation Sequencing of RNA on normal fibroblasts treated with RSV. To investigate whether the effects of RSV are mediated through SIRT1 we expanded the analysis to include SIRT1-knockdown fibroblasts. We identified the aspartoacylase (ASPA) gene, mutated in Canavan disease, to be strongly up-regulated by RSV in several cell lines, including Canavan disease fibroblasts. We further link RSV to the up-regulation of other genes involved in myelination including the glial specific transcription factors POU3F1, POU3F2, and myelin basic protein (MBP). We also observe a strong up-regulation by RSV of the riboflavin transporter gene SLC52a1. Mutations in SLC52a1 cause transient multiple acyl-CoA dehydrogenase deficiency (MADD). Our analysis of alternative splicing identified novel metabolically important genes affected by RSV, among which is particularly interesting the α subunit of the stimulatory G protein (Gsα), which regulates the cellular levels of cAMP through adenylyl cyclase. We conclude that in fibroblasts RSV stimulates the PGC-1α and p53 pathways, and up-regulates genes affecting the glucose metabolism, mitochondrial β-oxidation, and mitochondrial biogenesis. We further confirm that RSV might be a relevant treatment in the correction of FAO deficiencies and we suggest that treatment in other metabolic disorders including Canavan disease and MADD might be also beneficial.

<h4>Purpose</h4>A causal biomarker for acute respiratory distress syndrome (ARDS) could fuel precision therapy options. Plasma angiopoietin-2 (ANG2), a vascular permeability marker, is a strong candidate on the basis of experimental and observational evidence. We used genetic causal inference methods-Mendelian randomization and mediation-to infer potential effects of plasma ANG2.<h4>Methods</h4>We genotyped 703 septic subjects, measured ICU admission plasma ANG2, and performed a quantitative trait loci (QTL) analysis to determine variants in the ANGPT2 gene associated with plasma ANG2 (p < 0.005). We then used linear regression and post-estimation analysis to genetically predict plasma ANG2 and tested genetically predicted ANG2 for ARDS association using logistic regression. We estimated the proportion of the genetic effect explained by plasma ANG2 using mediation analysis.<h4>Results</h4>Plasma ANG2 was strongly associated with ARDS (OR 1.59 (95% CI 1.35, 1.88) per log). Five ANGPT2 variants were associated with ANG2 in European ancestry subjects (n = 404). Rs2442608C, the most extreme cis QTL (coefficient 0.22, 95% CI 0.09-0.36, p = 0.001), was associated with higher ARDS risk: adjusted OR 1.38 (95% CI 1.01, 1.87), p = 0.042. No significant QTL were identified in African ancestry subjects. Genetically predicted plasma ANG2 was associated with ARDS risk: adjusted OR 2.25 (95% CI 1.06-4.78), p = 0.035. Plasma ANG2 mediated 34% of the rs2442608C-related ARDS risk.<h4>Conclusions</h4>In septic European ancestry subjects, the strongest ANG2-determining ANGPT2 genetic variant is associated with higher ARDS risk. Plasma ANG2 may be a causal factor in ARDS development. Strategies to reduce plasma ANG2 warrant testing to prevent or treat sepsis-associated ARDS.

<h4>Background</h4>Previous studies have identified genetic variants associated with bronchopulmonary dysplasia (BPD) in extremely preterm infants. However, findings with genome-wide significance have been rare, and not replicated. We hypothesized that whole exome sequencing (WES) of premature subjects with extremely divergent phenotypic outcomes could facilitate the identification of genetic variants or gene networks contributing disease risk.<h4>Results</h4>The Prematurity and Respiratory Outcomes Program (PROP) recruited a cohort of > 765 extremely preterm infants for the identification of markers of respiratory morbidity. We completed WES on 146 PROP subjects (85 affected, 61 unaffected) representing extreme phenotypes of early respiratory morbidity. We tested for association between disease status and individual common variants, screened for rare variants exclusive to either affected or unaffected subjects, and tested the combined association of variants across gene loci. Pathway analysis was performed and disease-related expression patterns were assessed. Marginal association with BPD was observed for numerous common and rare variants. We identified 345 genes with variants unique to BPD-affected preterm subjects, and 292 genes with variants unique to our unaffected preterm subjects. Of these unique variants, 28 (19 in the affected cohort and 9 in unaffected cohort) replicate a prior WES study of BPD-associated variants. Pathway analysis of sets of variants, informed by disease-related gene expression, implicated protein kinase A, MAPK and Neuregulin/epidermal growth factor receptor signaling.<h4>Conclusions</h4>We identified novel genes and associated pathways that may play an important role in susceptibility/resilience for the development of lung disease in preterm infants.

Controlling the sources of Fe available to pathogens is one of the possible strategies that can be successfully used by novel antibacterial drugs. We focused our interest on the design of chelators to address <i>Mycobacterium avium</i> infections. Taking into account the molecular structure of mycobacterial siderophores and considering that new chelators must be able to compete for Fe(III), we selected ligands of the 3-hydroxy-4-pyridinone class to achieve our purpose. After choosing the type of chelating unit it was also our objective to design chelators that could be monitored inside the cell and for that reason we designed chelators that could be functionalized with fluorophores. We didn't realize at the time that the incorporation a fluorophore, to allow spectroscopic detection, would be so relevant for the antimycobacterial effect or to determine the affinity of the chelators towards biological membranes. From a biophysical perspective, this is a fascinating illustration of the fact that functionalization of a molecule with a particular label may lead to a change in its membrane permeation properties and result in a dramatic change in biological activity. For that reason we believe it is interesting to give a critical account of our entire work in this area and justify the statement "to label means to change". New perspectives regarding combined therapeutic approaches and the use of rhodamine B conjugates to target closely related problems such as bacterial resistance and biofilm production are also discussed.

Mucin glycoproteins on the ocular surface are rich in O-glycans and have important roles in the protection from physical, chemical and microbial impact. In this work, we have cultured human corneal and conjunctival epithelial cells to examine the glycosyltransferase activities that synthesize the O-glycans of mucins. The results indicate that ocular surface epithelial cells have active enzymes that synthesize O-glycans with sialylated core 1, Galβ1-3GalNAcα, and core 2, GlcNAcβ1-6(Galβ1-3)GalNAcα structures which corresponds to previous structural studies. Eye cells also have enzymes that synthesize complex N-glycans that are found on mucins. Results from treatment of eye cells with TNFα suggest that epithelial O-glycosylation changes in a dynamic fashion during inflammatory stimuli of the eye surface.

During violent criminal actions in which the perpetrator disposes of the victim's remains by burial, the analysis of insects and bacterial colonization patterns could be necessary for postmortem interval (PMI) estimation. Our research aimed to assess the decomposition process of buried rat carcasses from shallow graves (40 cm), the diversity and dynamics of insects and bacteria throughout the decomposition stages, and the environmental parameters' influence on these variations. The results provide further insight on decomposition in soil and contribute to a broader understanding of the factors involved in decomposition by qualitatively and quantitatively analysing the decomposer community (bacteria and insects). Additionally, two bacterial taxa, Enterococcus faecalis and Clostridium paraputrificum that were investigated for the first time as PMI indicators using quantitative polymerase chain reaction (qPCR) showed differential abundance over time, promising data for PMI estimation. The current study on the decomposition of buried rat carcasses in a natural environment will strengthen the current knowledge on decomposed remains from shallow graves and represents an effort to quantify insect and bacterial taxa as PMI estimators.

Osteoarthritis (OA) is a common joint disorder with increasing impact in an aging society. While genetic and transcriptomic analyses have revealed some genes and non-coding loci associated to OA, the pathogenesis remains incompletely understood. Chromatin profiling, which provides insight into gene regulation, has not been reported in OA mainly due to technical difficulties. Here, we employed Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq) to map the accessible chromatin landscape in articular knee cartilage of OA patients. We identified 109,215 accessible chromatin regions for cartilages, of which 71% were annotated as enhancers. By overlaying them with genetic and DNA methylation data, we have determined potential OA-relevant enhancers and their putative target genes. Furthermore, through integration with RNA-seq data, we characterized genes that are altered both at epigenomic and transcriptomic levels in OA. These genes are enriched in pathways regulating ossification and mesenchymal stem cell (MSC) differentiation. Consistently, the differentially accessible regions in OA are enriched for MSC-specific enhancers and motifs of transcription factor families involved in osteoblast differentiation. In conclusion, we demonstrate how direct chromatin profiling of clinical tissues can provide comprehensive epigenetic information for a disease and suggest candidate genes and enhancers of translational potential.

Genetic haemochromatosis (GH) is responsible for iron overload. Increased transferrin saturation (TSAT) has been associated with severe periodontitis, which is a chronic inflammatory disease affecting tissues surrounding the teeth and is related to dysbiosis of the subgingival microbiota. Because iron is essential for bacterial pathogens, alterations in iron homeostasis can drive dysbiosis. To unravel the relationships between serum iron biomarkers and the subgingival microbiota, we analysed samples from 66 GH patients. The co-occurrence analysis of the microbiota showed very different patterns according to TSAT. Healthy and periopathogenic bacterial clusters were found to compete in patients with normal TSAT (≤45%). However, significant correlations were found between TSAT and the proportions of Porphyromonas and Treponema, which are two genera that contain well-known periopathogenic species. In patients with high TSAT, the bacterial clusters exhibited no mutual exclusion. Increased iron bioavailability worsened periodontitis and promoted periopathogenic bacteria, such as Treponema. The radical changes in host-bacteria relationships and bacterial co-occurrence patterns according to the TSAT level also suggested a shift in the bacterial iron supply from transferrin to NTBI when TSAT exceeded 45%. Taken together, these results indicate that iron bioavailability in biological fluids is part of the equilibrium between the host and its microbiota.

Circadian rhythms influence physiological processes from sleep-wake cycles to body temperature and are controlled by highly conserved cycling molecules. Although the mechanistic basis of the circadian clock has been known for decades, the extent to which circadian rhythms vary in nature and the underlying genetic basis for that variation is not well understood. We measured circadian period (Ʈ) and rhythmicity index in the Drosophila Genetic Reference Panel (DGRP) and observed extensive genetic variation in both. Seven DGRP lines had sexually dimorphic arrhythmicity and one line had an exceptionally long Ʈ. Genome-wide analyses identified 584 polymorphisms in 268 genes. We observed differences among transcripts for nine genes predicted to interact among themselves and canonical clock genes in the long period line and a control. Mutations/RNAi knockdown targeting these genes also affected circadian behavior. Our observations reveal that complex genetic interactions influence high levels of variation in circadian phenotypes.

The reactivity of 1-hydroxybenzoyl triazole (HOBt) esters with the carboxylate functionality present in peptides is demonstrated in the gas phase with a doubly deprotonated dianion. The reaction forms an anhydride linkage at the carboxylate site. Upon ion trap collisional-induced dissociation (CID) of the modified peptide, the resulting spectrum shows a nominal loss of the mass of the reagent and a water molecule. Analogous phenomenology was also noted for model peptide cations that likely contain zwitterionic/salt-bridged motifs in reactions with a negatively charged HOBt ester. Control experiments indicate that a carboxylate group is the likely reactive site, rather than other possible nucleophilic sites present in the peptide. These observations suggest that HOBt ester chemistry may be used as a chemical probe for the presence and location of carboxylate groups in net positively charged polypeptide ions. As an illustration, deprotonated sulfobenzoyl HOBt was reacted with the [M+7H]⁷⁺ ion of ubiquitin. The ion was shown to react with the reagent and CID of the covalent reaction product yielded an abundant [M+6H-H₂O]⁶⁺ ion. Comparison of the CID product ion spectrum of this ion with that of the water loss product generated from CID of the unmodified [M+6H]⁶⁺ ion revealed the glutamic acid at residue 64 as a reactive site, suggesting that it is present in the deprotonated form. Graphical Abstract ᅟ.

The application of next-generation sequencing (NGS) technology in clinical diagnostics should proceed with care. We have evaluated the clinical validity of two commercially available RNA fusion panels, the TruSight RNA fusion panel (Illumina) and FusionPlex Pan-Heme Kit (ArcherDx), to detect recurrent translocations in hematologic malignancies. Twenty-four bone marrow samples taken at the initial diagnosis of patients with acute leukemia and chronic myeloid leukemia were included. To assess the limit of detection, serial dilutions of BCR-ABL1 (e1a2)-positive RNAs were prepared using a commercial reference material. Both NGS panels detected 19 cases with recurrent translocations identified with RT-PCR, as well as a case with KMT2A-AFF1 with false-negative results on RT-PCR. Two rare translocations, DDX3X-MLLT10 and NUP98-HOXC13, were additionally identified using NGS panels. The detection limit ranged from 10^-1 to 10^-2, which was not satisfactory for samples with low tumor burden. To conclude, RNA fusion panels were suitable for the initial diagnosis; however, for follow-up samples, conventional RT-PCR should be selected.

Iron chelation has been introduced as a new therapeutic concept for the treatment of neurodegenerative diseases with features of iron overload. At difference with iron chelators used in systemic diseases, effective chelators for the treatment of neurodegenerative diseases must cross the blood⁻brain barrier. Given the promissory but still inconclusive results obtained in clinical trials of iron chelation therapy, it is reasonable to postulate that new compounds with properties that extend beyond chelation should significantly improve these results. Desirable properties of a new generation of chelators include mitochondrial destination, the center of iron-reactive oxygen species interaction, and the ability to quench free radicals produced by the Fenton reaction. In addition, these chelators should have moderate iron binding affinity, sufficient to chelate excessive increments of the labile iron pool, estimated in the micromolar range, but not high enough to disrupt physiological iron homeostasis. Moreover, candidate chelators should have selectivity for the targeted neuronal type, to lessen unwanted secondary effects during long-term treatment. Here, on the basis of a number of clinical trials, we discuss critically the current situation of iron chelation therapy for the treatment of neurodegenerative diseases with an iron accumulation component. The list includes Parkinson's disease, Friedreich's ataxia, pantothenate kinase-associated neurodegeneration, Huntington disease and Alzheimer's disease. We also review the upsurge of new multifunctional iron chelators that in the future may replace the conventional types as therapeutic agents for the treatment of neurodegenerative diseases.

<h4>Objective</h4>Obesity is a complex disorder involving many genetic and environmental factors that are required to maintain energy homeostasis. While studies in human populations have led to significant progress in the generation of an obesity gene map and broadened our understanding of the genetic basis of common obesity, there is still a large portion of heritability and etiology that remains unknown. Here, we have used the genetically tractable fruit fly, Drosophila melanogaster, to identify genes/pathways that function in the nervous system to regulate energy balance.<h4>Methods</h4>We performed an in vivo RNAi screen in Drosophila neurons and assayed for obese or lean phenotypes by measuring changes in levels of stored fats (in the form of triacylglycerides or TAG). Three rounds of screening were performed to verify the reproducibility and specificity of the adiposity phenotypes. Genes that produced >25% increase in TAG (206 in total) underwent a second round of screening to verify their effect on TAG levels by retesting the same RNAi line to validate the phenotype. All remaining hits were screened a third time by testing the TAG levels of additional RNAi lines against the genes of interest to rule out any off-target effects.<h4>Results</h4>We identified 24 genes including 20 genes that have not been previously associated with energy homeostasis. One identified hit, Diacylglycerol kinase (Dgk), has mammalian homologues that have been implicated in genome-wide association studies for metabolic defects. Downregulation of neuronal Dgk levels increases TAG and carbohydrate levels and these phenotypes can be recapitulated by reducing Dgk levels specifically within the insulin-producing cells that secrete Drosophila insulin-like peptides (dILPs). Conversely, overexpression of kinase-dead Dgk, but not wild-type, decreased circulating dILP2 and dILP5 levels resulting in lower insulin signalling activity. Despite having higher circulating dILP levels, Dgk RNAi flies have decreased pathway activity suggesting that they are insulin-resistant.<h4>Conclusion</h4>Altogether, we have identified several genes that act within the CNS to regulate energy homeostasis. One of these, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila.

We have explored orally effective thyrotropin-releasing hormone (TRH) mimetics, showing oral bioavailability and brain penetration by structure-activity relationship (SAR) study on the basis of in vivo antagonistic activity on reserpine-induced hypothermia in mice. By primary screening of the synthesized TRH mimetics, we found a novel TRH mimetic: l-pyroglutamyl-[3-(thiazol-4-yl)-l-alanyl]-l-prolinamide with a high central nervous system effect compared with TRH as a lead compound. Further SAR optimization studies of this lead compound led to discovery of a novel orally effective TRH mimetic: 1-{<i>N</i>-[(4<i>S</i>,5<i>S</i>)-(5-methyl-2-oxooxazolidine-4-yl)carbonyl]-3-(thiazol-4-yl)-l-alanyl}-(2<i>R</i>)-2-methylpyrrolidine trihydrate (rovatirelin hydrate), which was selected as a candidate for clinical trials.

Artemisinin-estrogen hybrids were for the first time both synthesized and investigated for their <i>in vitro</i> biological activity against malaria parasites (<i>Plasmodium falciparum</i> 3D7), human cytomegalovirus (HCMV), and a panel of human malignant cells of gynecological origin containing breast (MCF7, MDA-MB-231, MDA-MB-361, T47D) and cervical tumor cell lines (HeLa, SiHa, C33A). In terms of antimalarial efficacy, hybrid <b>8</b> (EC₅₀ = 3.8 nM) was about two times more active than its parent compound artesunic acid (<b>7</b>) (EC₅₀ = 8.9 nM) as well as the standard drug chloroquine (EC₅₀ = 9.8 nM) and was, therefore, comparable to the clinically used dihydroartemisinin (<b>6</b>) (EC₅₀ = 2.4 nM). Furthermore, hybrids <b>9</b>-<b>12</b> showed a strong antiviral effect with EC₅₀ values in the submicromolar range (0.22-0.38 μM) and thus possess profoundly stronger anti-HCMV activity (approximately factor 25) than the parent compound artesunic acid (<b>7</b>) (EC₅₀ = 5.41 μM). These compounds also exerted a higher <i>in vitro</i> anti-HCMV efficacy than ganciclovir used as the standard of current antiviral treatment. In addition, hybrids <b>8</b>-<b>12</b> elicited substantially more pronounced growth inhibiting action on all cancer cell lines than their parent compounds and the reference drug cisplatin. The most potent agent, hybrid <b>12</b>, exhibited submicromolar EC₅₀ values (0.15-0.93 μM) against breast cancer and C33A cell lines.

Recent years have seen increased interest in psychopathologies related to trauma exposure. Specifically, there has been a growing awareness to posttraumatic stress disorder (PTSD) in part due to terrorism, climate change-associated natural disasters, the global refugee crisis, and increased violence in overpopulated urban areas. However, notwithstanding the increased awareness to the disorder, the increasing number of patients, and the devastating impact on the lives of patients and their families, the efficacy of available treatments remains limited and highly unsatisfactory. A major scientific effort is therefore devoted to unravel the neural mechanisms underlying PTSD with the aim of paving the way to developing novel or improved treatment approaches and drugs to treat PTSD. One of the major scientific tools used to gain insight into understanding physiological and neuronal mechanisms underlying diseases and for treatment development is the use of animal models of human diseases. While much progress has been made using these models in understanding mechanisms of conditioned fear and fear memory, the gained knowledge has not yet led to better treatment options for PTSD patients. This poor translational outcome has already led some scientists and pharmaceutical companies, who do not in general hold opinions against animal models, to propose that those models should be abandoned. Here, we critically examine aspects of animal models of PTSD that may have contributed to the relative lack of translatability, including the focus on the exposure to trauma, overlooking individual and sex differences, and the contribution of risk factors. Based on findings from recent years, we propose research-based modifications that we believe are required in order to overcome some of the shortcomings of previous practice. These modifications include the usage of animal models of PTSD which incorporate risk factors and of the behavioral profiling analysis of individuals in a sample. These modifications are aimed to address factors such as individual predisposition and resilience, thus taking into consideration the fact that only a fraction of individuals exposed to trauma develop PTSD. We suggest that with an appropriate shift of practice, animal models are not only a valuable tool to enhance our understanding of fear and memory processes, but could serve as effective platforms for understanding PTSD, for PTSD drug development and drug testing.

A series of eight novel phosphatidylcholines containing cinnamic or 3-methoxycinnamic acids (3a-b, 5a-b, 9a-b, 10a-b) at <i>sn</i>-1 and/or <i>sn</i>-2 positions were synthesized and tested for their antiproliferative activity in an <i>in vitro</i> model against representative six human cancer cell lines (MV4-11, A549, MCF-7, LoVo, LoVo/DX, HepG2) and a normal cell line BALB/3T3. The structures of the new compounds were confirmed by spectral analysis. Biological evaluation revealed that all the tested conjugates exhibited higher antitumor activity than the corresponding free aromatic acids. Compounds 3b and 9b turned out to be the most active, with IC₅₀ values of 32.1 and 30.5 μM against the LoVo/DX and MV4-11 cell lines, respectively. Studies of the mechanism of the antitumor action were carried out for 1-palmitoyl-2-cinnamoyl-<i>sn</i>-glycero-3-phosphocholine (5a), and it was shown to be active toward almost all the tested types of cancer cells, showing that this compound could effectively arrest the cell cycle in G2/M and decrease the mitochondrial membrane potential of leukemia MV4-11 cells. The obtained results proved that the strategy of the incorporation of cinnamic and 3-methoxycinnamic acids into phospholipids could expand their potential application in industry, as well as could improve their antiproliferative activity and selectivity toward cancer cell lines.

This review focuses on the pathways that regulate lysosome biogenesis and that are implicated in numerous degenerative storage diseases, including lysosomal storage disorders and late-onset neurodegenerative diseases. Lysosomal proteins are synthesized in the endoplasmic reticulum and trafficked to the endolysosomal system through the secretory route. Several receptors have been characterized that execute post-Golgi trafficking of lysosomal proteins. Some of them recognize their cargo proteins based on specific amino acid signatures, others based on a particular glycan modification that is exclusively found on lysosomal proteins. Nearly all receptors serving lysosome biogenesis are under the transcriptional control of transcription factor EB (TFEB), a master regulator of the lysosomal system. TFEB coordinates the expression of lysosomal hydrolases, lysosomal membrane proteins, and autophagy proteins in response to pathways sensing lysosomal stress and the nutritional conditions of the cell among other stimuli. TFEB is primed for activation in lysosomal storage disorders but surprisingly its function is impaired in some late-onset neurodegenerative storage diseases like Alzheimer's and Parkinson's, because of specific detrimental interactions that limit TFEB expression or activation. Thus, disrupted TFEB function presumably plays a role in the pathogenesis of these diseases. Multiple studies in animal models of degenerative storage diseases have shown that exogenous expression of TFEB and pharmacological activation of endogenous TFEB attenuate disease phenotypes. These results highlight TFEB-mediated enhancement of lysosomal biogenesis and function as a candidate strategy to counteract the progression of these diseases. This article is part of the Special Issue "Lysosomal Storage Disorders".

Mounting attention has been focused on defects in macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemia. TFEB (transcription factor EB)-mediated induction of ALP has been recently considered as the common mechanism in ameliorating the pathological lesion of myocardial ischemia and neurodegenerative diseases. Here we explored the vital role of TFEB in permanent middle cerebral artery occlusion (pMCAO)-mediated dysfunction of ALP and ischemic insult in rats. The results showed that ALP function was first enhanced in the early stage of the ischemic process, especially in neurons of the cortex, and this was accompanied by increased TFEB expression and translocation to the nucleus, which was mediated at least in part through activation by PPP3/calcineurin. At the later stages of ischemia, a gradual decrease in the level of nuclear TFEB was coupled with a progressive decline in lysosomal activity, accumulation of autophagosomes and autophagy substrates, and exacerbation of the ischemic injury. Notably, neuron-specific overexpression of TFEB significantly enhanced ALP function and rescued the ischemic damage, starting as early as 6 h and even lasting to 48 h after ischemia. Furthermore, neuron-specific knockdown of TFEB markedly reversed the activation of ALP and further aggravated the neurological deficits and ischemic outcome at the early stage of pMCAO. These results highlight neuronal-targeted TFEB as one of the key players in the pMCAO-mediated dysfunction of ALP and ischemic injury, and identify TFEB as a promising target for therapies aimed at neuroprotection in cerebral ischemia. Abbreviations: AAV, adeno-associated virus; AIF1/IBA1, allograft inflammatory factor 1; ALP, autophagy-lysosomal pathway; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; CsA, cyclosporin A; GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; MAP2, microtubule-associated protein 2; mNSS, modified Neurological Severity Score; MTOR, mechanistic target of rapamycin kinase; OGD, oxygen and glucose deprivation; pMCAO, permanent middle cerebral artery occlusion; RBFOX3/NeuN, RNA binding fox-1 homolog 3; SQSTM1, sequestosome1; TFEB, transcription factor EB; TTC, 2,3,5-triphenyltetrazolium chloride.

Nanoparticles often only exploit the upregulation of a receptor on cancer cells to enhance intratumoral deposition of therapeutic and imaging agents. However, a single targeting moiety assumes that a tumor is homogenous and static. Tumoral microenvironments are both heterogenous and dynamic, often displaying variable spatial and temporal expression of targetable receptors throughout disease progression. Here, we evaluated the in vivo performance of an iron oxide nanoparticle in terms of targeting and imaging of orthotropic mouse models of aggressive breast tumors. The nanoparticle, a multi-component nanochain, was comprised of 3-5 iron oxide nanoparticles chemically linked in a linear chain. The nanoparticle's surface was decorated with two types of ligands each targeting two different upregulated biomarkers on the tumor endothelium, P-selectin and fibronectin. The nanochain exhibited improved tumor deposition not only through vascular targeting but also through its elongated structure. A single-ligand nanochain exhibited a ~2.5-fold higher intratumoral deposition than a spherical nanoparticle variant. Furthermore, the dual-ligand nanochain exhibited higher consistency in generating detectable MR signals compared to a single-ligand nanochain. Using a 7T MRI, the dual-ligand nanochains exhibited highly detectable MR signal within 3h after injection in two different animal models of breast cancer.

<h4>Background</h4>European studies identified the C9orf72 repeat expansion as the most frequent genetic alteration in patients with Huntington disease (HD)-like phenotypes but negative HD genetic testing.<h4>Objective</h4>To investigate C9orf72 repeat expansion frequency in individuals tested for HD in a North American tertiary referral laboratory.<h4>Methods</h4>Three hundred and seventy-three cases (115 positive and 258 negative for HD) were evaluated by genotyping PCR, with follow-up Southern blot and 5' repeat methylation status assessment by combined repeat-primed and methylation-specific PCR in a subset.<h4>Results</h4>Three cases (all HD-negative) tested positive: 2 had > 2,000 repeats and were methylated, 1 had 80-100 repeats and was unmethylated. Two cases (1 HD-positive and 1 HD-negative) had intermediate alleles (20-29 repeats) and were unmethylated. The remaining 368 cases were negative (< 20 repeats). C9orf72 repeat expansion was absent in patients with HD and was identified in a small subset (1.2%) of patients with negative HD genetic testing.<h4>Conclusion</h4>These findings suggest that C9orf72 repeat expansion does not coexist with HTT repeat expansion and that C9orf72 repeat expansion testing is unnecessary for patients with HD. In addition, C9orf72 evaluation may be considered for individuals negative for HD genetic testing. Similar to in previous studies, methylation of C9orf72 repeat expansion was limited to large expansions.

Long non-coding RNAs (lncRNAs) are an emerging class of regulatory RNA that may be implicated in psychiatric disorders. Here we performed RNA-sequencing in the rostral anterior cingulate cortex of 26 depressed suicides and 24 matched controls. We first performed differential lncRNA expression analysis, and then conducted Weighted Gene Co-expression Network Analysis (WGCNA) to identify co-expression modules associating with depression and suicide. We identified 23 differentially expressed lncRNAs (FDR < 0.1) as well as their differentially expressed overlapping and antisense protein-coding genes. Several of these overlapping or antisense genes were associated with interferon signaling, which is a component of the innate immune response. Using WGCNA, we identified modules of highly co-expressed genes associated with depression and suicide and found protein-coding genes highly connected to differentially expressed lncRNAs within these modules. These protein-coding genes were located distal to their associated lncRNAs and were found to be part of several GO terms enriched in the significant modules, which include: cytoskeleton organization, plasma membrane, cell adhesion, nucleus, DNA-binding, and regulation of dendrite development and morphology. Altogether, we report that lncRNAs are differentially expressed in the brains of depressed individuals who died by suicide and may represent regulators of important molecular functions and biological processes.

The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-β₂m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.

TROY is a component of the Nogo receptor complex and plays the key role in neuronal survival, migration, and differentiation. Here, we show the up-regulation of TROY in human glioma tissues and cells. Inhibition of TROY expression slowed glioma development in vivo and in vitro. Raf kinase inhibitor (RKIP) was found to interact with TROY. The physical interaction of TROY/RKIP was confirmed via co-immunoprecipitation (co-IP) assays. Furthermore, we found that the TROY/RKIP interaction was enhanced by fetal bovine serum (FBS) exposure, and TROY knockdown also led to down-regulation of NF-κB. Finally, disruption of the TROY/RKIP interaction using the TAT-TROY (234-371 aa) protein reduced the glioma development in xenografted mice. This suggests the TROY/RKIP interaction is a potential target for therapy of gliomas.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. <i>Drosophila</i> studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.

Bone homeostasis is a complex process, wherein osteoclasts resorb bone and osteoblasts produce new bone tissue. For the maintenance of skeletal integrity, this sequence has to be tightly regulated and orchestrated. Iron overload as well as iron deficiency disrupt the delicate balance between bone destruction and production, via influencing osteoclast and osteoblast differentiation as well as activity. Iron overload as well as iron deficiency are accompanied by weakened bones, suggesting that balanced bone homeostasis requires optimal-not too low, not too high-iron levels. The goal of this review is to summarize our current knowledge about how imbalanced iron influence skeletal health. Better understanding of this complex process may help the development of novel therapeutic approaches to deal with the pathologic effects of altered iron levels on bone.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α₁-subunit of the voltage-gated Ca_V2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed Ca_V2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Considerable evidence suggests loss-of-function mutations in the chromatin remodeler CHD2 contribute to a broad spectrum of human neurodevelopmental disorders. However, it is unknown how CHD2 mutations lead to impaired brain function. Here we report mice with heterozygous mutations in Chd2 exhibit deficits in neuron proliferation and a shift in neuronal excitability that included divergent changes in excitatory and inhibitory synaptic function. Further in vivo experiments show that Chd2^+/- mice displayed aberrant cortical rhythmogenesis and severe deficits in long-term memory, consistent with phenotypes observed in humans. We identified broad, age-dependent transcriptional changes in Chd2^+/- mice, including alterations in neurogenesis, synaptic transmission, and disease-related genes. Deficits in interneuron density and memory caused by Chd2^+/- were reproduced by Chd2 mutation restricted to a subset of inhibitory neurons and corrected by interneuron transplantation. Our results provide initial insight into how Chd2 haploinsufficiency leads to aberrant cortical network function and impaired memory.

Gaucher disease (GD) is an autosomal recessive condition that results from a deficiency of the enzyme β-glucocerebrosidase. The increased risk of primary parkinsonism symptoms among individuals affected with GD and carriers for the disorder is well-documented in the literature. However, these risks and case reports often reflect patients with classical Parkinson's disease (PD) symptoms. We report a patient with GD type 1 who was diagnosed with corticobasal syndrome (CBS), a clinical atypical parkinsonism diagnosis, in his sixth decade of life. Our case highlights the need to consider forms of atypical parkinsonism such as CBS in addition to PD in the differential diagnosis of cognitive and motor changes in patients with GD type 1. We also recommend careful assessment and routine monitoring of cognition, mood, behavior, sleep patterns, olfaction, and memory in patients with GD type 1 to identify early symptoms indicative of neurological involvement.

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in <i>Drosophila</i> and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar-phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in <i>Drosophila</i> when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

Hepatocellular carcinoma (HCC), accounting for approximately 90% of liver cancer, is the most lethal malignant tumors in the world. Large amount of evidence indicate that microRNAs (miRNAs) contribute to the tumorigenesis and progression of HCC. Among them, miR-376c-3p was recently identified as a tumor-related miRNA and is up-regulated in HBV-related HCC. But, the clinical significance of miR-376c-3p and its biological function in HCC progression are still unclear. Here, we confirmed that miR-376c-3p expression level in HCC was markedly higher than that in noncancerous tissues. Up-regulation of miR-376c-3p was detected in four different HCC cell lines. High miR-376c-3p expression correlated with poor prognostic features, such as large tumor size and venous infiltration. Follow-up data indicated that high miR-376c-3p level evidently correlated with poor clinical outcomes of HCC patients. Moreover, knockdown of miR-376c-3p repressed HCC cell growth, migration and invasion <i>in vitro</i>. miR-376c-3p overexpression facilitated these malignant behaviors of Bel-7402 cells. Mechanistically, miR-376c-3p posttranscriptionally repressed ARID2 expression by directly interacting with its 3'-UTR. Furthermore, an obvious negative correlation between miR-376c-3p and ARID2 mRNA expression in HCC tissues was confirmed. Notably, miR-376c-3p knockdown suppressed HCC growth and metastasis in nude mice. Gain-of-function experiments showed that ARID2 inhibited cell growth and mobility of Hep3B cells. Subsequently, ARID2 knockdown rescued miR-376c-3p silencing attenuated Hep3B cell proliferation and mobility. Our results suggest that miR-376c-3p exerts an oncogenic role in HCC progression.

We have developed a sheet-like pH imaging sensor based on a flexible and physically adhesive polymer thin film (referred to as a "pH sensing nanosheet"). The pH sensing nanosheet was composed of two films: one is a pH-sensitive layer-by-layer (LbL) film constructed from fluorescein-conjugated poly(acrylic acid) and poly(allylamine hydrochloride) and the other is a pH-insensitive film made from Nile red-embedded poly(d,l-lactic acid). The pH sensing nanosheet enabled the ratiometric imaging of pH changes in a leaf (500 × 500 μm²), namely the apoplastic ion milieu responding to an external NaCl stress. It was successfully mapped out that the alkalization of the leaf apoplast spread from the leaf base to the tip at 20 min after the stimulation and the pH value increased up to approximately pH 6.3 from less than pH 4.5 within 60 min when a 100 mM NaCl aqueous solution was added. The pH sensing nanosheet should be useful for energy metabolic mapping in tissue biology.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.

<h4>Background</h4>Gestational hypothyroidism (G-HypoT) is one of the most common thyroid diseases in pregnant women. Human milk, which closely links the mother with infant, is an important factor to the infant health. Here, we analyzed the colostrum whey proteome of women with or without G-HypoT.<h4>Methods and results</h4>Using high-mass accuracy and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), 1055 proteins were identified. Tandem Mass Tags (TMT) analysis identified differentially expressed proteins between G-HypoT and non-G-HypoT mothers. Of 44 proteins identified, 15 proteins were significantly increased in G-HypoT colostrum whey, while 29 were significantly decreased. Analysis revealed that enzymes involved in carbohydrate metabolism, and that reflect the metabolic activities in breastfeeding women, including fructose-1, 6-bisphosphatase 1, phosphoglycerate mutase 1 were down-regulated. Cell structural proteins, biomarkers of mammary integrity development, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin were lower in G-HypoT colostrum whey. However, immune protein fragments like Ig gamma-3 chain C region increased in G-HypoT colostrum whey.<h4>Conclusion</h4>These results implied that G-HypoT may changed human colostrum whey protein in composition level, decreasing levels of metabolic proteins and cell-structure proteins, while increasing levels of immune-related proteins, which may compromise or reflect mothers' and infants' health.

A prominent organizational feature of bacterial chromosomes was revealed by Hi-C experiments, indicating anomalously high contacts between the left and right chromosomal arms. These long-range contacts have been attributed to various nucleoid-associated proteins, including the ATPase Structural Maintenance of Chromosomes (SMC) condensin. Although the molecular structure of these ATPases has been mapped in detail, it still remains unclear by which physical mechanisms they collectively generate long-range chromosomal contacts. Here, we develop a computational model that captures the subtle interplay between molecular-scale activity of slip-links and large-scale chromosome organization. We first consider a scenario in which the ATPase activity of slip-links regulates their DNA-recruitment near the origin of replication, while the slip-link dynamics is assumed to be diffusive. We find that such diffusive slip-links can collectively organize the entire chromosome into a state with aligned arms, but not within physiological constraints. However, slip-links that include motor activity are far more effective at organizing the entire chromosome over all length-scales. The persistence of motor slip-links at physiological densities can generate large, nested loops and drive them into the bulk of the DNA. Finally, our model with motor slip-links can quantitatively account for the rapid arm-arm alignment of chromosomal arms observed <i>in vivo</i>.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset-accessible through the Beat AML data viewer (Vizome)-that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.

Bioactive alloplastic materials, like beta-tricalcium phosphate (β-TCP) and calcium sulfate (CS), have been extensively researched and are currently used in orthopedic and dental bone regenerative procedures. The purpose of this study was to compare the performance of EthOss versus a bovine xenograft and spontaneous healing. The grafting materials were implanted in standardized 8 mm circular bicortical bone defects in rabbit calvariae. A third similar defect in each animal was left empty for natural healing. Six male rabbits were used. After eight weeks of healing, the animals were euthanized and the bone tissue was analyzed using histology and micro-computed tomography (micro-CT). Defects treated with β-TCP/CS showed the greatest bone regeneration and graft resorption, although differences between groups were not statistically significant. At sites that healed spontaneously, the trabecular number was lower (<i>p</i> < 0.05) and trabecular separation was higher (<i>p</i> < 0.05), compared to sites treated with β-TCP/CS or xenograft. Trabecular thickness was higher at sites treated with the bovine xenograft (<i>p</i> < 0.05) compared to sites filled with β-TCP/CS or sites that healed spontaneously. In conclusion, the novel β-TCP/CS grafting material performed well as a bioactive and biomimetic alloplastic bone substitute when used in cranial defects in this animal model.

Two synthetic protocols for the introduction of fluorine atoms into resorcinarene-based cavitands, at the lower and upper rim, respectively, are reported. Cavitand <b>1</b>, bearing four fluorocarbon tails, and cavitand <b>2</b>, which presents a fluorine atom on the <i>para</i> position of a diester phosphonate phenyl substituent, were synthesized and their complexation abilities toward the model guest sarcosine methyl ester hydrochloride were evaluated via NMR titration experiments. The effect of complexation on the ¹⁹F NMR resonance of the probe is evident only in the case of cavitand <b>2</b>, where the inset of the cation-dipole and H-bonding interactions between the P=O bridges and the guest is reflected in a sizable downfield shift of the fluorine probe.

Urinary or serum uric acid concentration is an indicator of chronic kidney condition. An increase in uric acid concentration may indicate renal dysfunction. Reliable instantaneous detection of uric acid without requiring sophisticated laboratory and analytical instrumentation, such as: chromatographic and spectrophotometric methods, would be invaluable for patients with renal complication. This paper reports the early development of a simple, low-cost, instantaneous and user-friendly paper based diagnostic device (PAD) for the qualitative and quantitative detection of uric acid in urine. A colorimetric detection technique was developed based on the intensity of Prussian blue color formation on paper in presence of uric acid; the reaction rate of corresponding chemical reactions on paper surface was also studied. Based on the colorimetric signal produced on paper surface, a calibration curve was developed to detect unknown concentration of uric acid in urine. The effect of temperature on formation of color signal on paper surface was also analyzed. In this study, estimation of urinary uric acid using MATLAB coding on a windows platform was demonstrated as the use of software application and digital diagnostics. This paper-based technique is faster and less expensive compared to traditional detection techniques. The paper-based diagnostic can be integrated with a camera of smart phone, tablet computer or laptop and an image processing application (using windows/android/IOS platform) as a part of digital diagnostics. Therefore, with proper calibration, the paper-based technique can be compatible and economical to the sophisticated detection techniques used to detect urinary uric acid.

Data herein describe the quantitative changes in the plasma proteome in chickens challenged with lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate the host innate immune system obtained by shotgun quantitative proteomic tandem mass tags approach using high-resolution Orbitrap technology. Statistical and bioinformatic analyses were performed to specify the effect of bacterial endotoxin. Plasma from chicken (N=6) challenged with <i>Escherichia coli</i> (LPS) (2 mg/kg body weight) was collected pre (0 h) and at 12, 24, 48, and 72 h post injection along with plasma from a control group (N=6) challenged with sterile saline. Protein identification and relative quantification were performed using Proteome Discoverer, and data were analysed using R. Gene Ontology terms were analysed by the Cytoscape application ClueGO based on <i>Gallus gallus</i> GO Biological Process database, and refined by REVIGO. Absolute quantification of several acute phase proteins, e.g. alpha-1-acid glycoprotein (AGP), serum amyloid A (SAA) and ovotrensferrin (OVT) was performed by immunoassays to validate the LC-MS results. The data contained within this article are directly related to our research article"<i>Quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin"</i> [1]. The raw mass spectrometric data generated in this study were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009399 (http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD009399).

No abstract available.

Accumulating evidence suggests that misfolded MAPT (microtubule associated protein tau), the main component of neurofibrillary tangles in tauopathies, is subject to degradation by the autophagy-lysosomal pathway. Selective autophagy is a subtype of macroautophagy that requires cargo receptors, such as OPTN (optineurin) or SQSTM1, to recognize specific targets for their sequestration within the autophagosome and their eventual degradation by the lysosome, although their roles in targeting distinct MAPT species have not been fully investigated. Using cargo receptor knockout cell lines and a seeding-based cellular assay in which neurofibrillary tangle pathology can be modeled in vitro, we reveal that while OPTN primarily targets soluble MAPT expressed in physiological conditions, SQSTM1 predominantly degrades insoluble but not soluble mutant MAPT. Endogenous SQSTM1 colocalizes with misfolded and aggregated MAPT species in vitro and in vivo, and both this colocalization and its function in MAPT clearance require both the LC3-interacting region (LIR) motif and also the PB1 self-polymerization domain of SQSTM1. Further, pathogenic MAPT accumulation reduces basal macroautophagy/autophagy in vitro and is associated with a compensatory upregulation of the lysosomal pathway in vivo. Finally, increased expression of SQSTM1 in MAPT transgenic mouse brains ameliorates MAPT pathology and prion-like spreading. Our results uncover distinct properties of selective autophagy receptors in targeting different MAPT species, implicate compromised autophagy as a potential underlying factor in mutant MAPT deposition, and demonstrate a potent and specific role of SQSTM1 in targeted clearance of pathogenic MAPT, through which it blocks neurofibrillary tangle accumulation and pathological spreading. Abbreviations: AAV: adeno-associated virus; AD: Alzheimer disease; ALP: autophagy-lysosomal pathway; ALS: amyotrophic lateral sclerosis; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; FTD: frontotemporal dementias; HD: Huntington disease; HTT: huntingtin; LIR: LC3-interacting region; NBR1: autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NFTs: neurofibrillary tangles; MAPT: microtubule associated protein tau; OPTN: optineurin; p-MAPT: hyperphosphorylated MAPT; PFA: paraformaldehyde; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1 Tax1: binding protein 1; ThioS: thioflavin-S; UBA: ubiquitin-associated.

Individuals with psychiatric disorders have elevated rates of autoimmune comorbidity and altered immune signaling. It is unclear whether these altered immunological states have a shared genetic basis with those psychiatric disorders. The present study sought to use existing summary-level data from previous genome-wide association studies to determine if commonly varying single nucleotide polymorphisms are shared between psychiatric and immune-related phenotypes. We estimated heritability and examined pair-wise genetic correlations using the linkage disequilibrium score regression (LDSC) and heritability estimation from summary statistics methods. Using LDSC, we observed significant genetic correlations between immune-related disorders and several psychiatric disorders, including anorexia nervosa, attention deficit-hyperactivity disorder, bipolar disorder, major depression, obsessive compulsive disorder, schizophrenia, smoking behavior, and Tourette syndrome. Loci significantly mediating genetic correlations were identified for schizophrenia when analytically paired with Crohn's disease, primary biliary cirrhosis, systemic lupus erythematosus, and ulcerative colitis. We report significantly correlated loci and highlight those containing genome-wide associations and candidate genes for respective disorders. We also used the LDSC method to characterize genetic correlations among the immune-related phenotypes. We discuss our findings in the context of relevant genetic and epidemiological literature, as well as the limitations and caveats of the study.

Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core.

<h4>Background</h4>Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil.<h4>Results</h4>Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria.<h4>Conclusions</h4>Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

<h4>Background</h4>Sarcomas account for less than 1% of primary breast cancers, and breast angiosarcomas are responsible for only 0.05% of all breast malignancies. The male breast has the same potential for malignant transformation as the female breast. However, due to anatomical differences in the breast and the low incidence of angiosarcoma, it is difficult to determine how male breasts can be affected by this type of tumor.<h4>Case presentation</h4>A 36-year-old male patient was admitted to the hospital with a palpable lump in his right breast. Lymphadenopathy was negative. Ultrasonography showed a hypoechoic mass with partially defined contours, measuring 4.0 × 3.0 cm, with muscle infiltration. Histological examination revealed a malignant tumor. Radical mastectomy was then performed with clear surgical margins. The patient began chemotherapy with paclitaxel. Following the second cycle of chemotherapy, he presented with headache and seizures due to a frontal lobe metastasis. Twenty days after the onset of neurological symptoms, the patient died.<h4>Conclusions</h4>Primary angiosarcomas of the male breast are extremely rare. This is the sixth case published in the literature. It is in agreement with other studies in the literature concerning clinical presentation and poor prognosis. Treatment consists in surgical removal of the tumor with clear margins and without axillary lymphadenectomy.

<h4>Purpose</h4>Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of <i>ex vivo</i>-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor.<h4>Experimental design</h4>The role of PIM kinases in T cells was studied either by genetic ablation (PIM1^-/-PIM2^-/-PIM3^-/-) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both.<h4>Results</h4>With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination.<h4>Conclusions</h4>This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Chromatin structure and function is regulated by reader proteins recognizing histone modifications and/or histone variants. We recently identified that PWWP2A tightly binds to H2A.Z-containing nucleosomes and is involved in mitotic progression and cranial-facial development. Here, using in vitro assays, we show that distinct domains of PWWP2A mediate binding to free linker DNA as well as H3K36me3 nucleosomes. In vivo, PWWP2A strongly recognizes H2A.Z-containing regulatory regions and weakly binds H3K36me3-containing gene bodies. Further, PWWP2A binds to an MTA1-specific subcomplex of the NuRD complex (M1HR), which consists solely of MTA1, HDAC1, and RBBP4/7, and excludes CHD, GATAD2 and MBD proteins. Depletion of PWWP2A leads to an increase of acetylation levels on H3K27 as well as H2A.Z, presumably by impaired chromatin recruitment of M1HR. Thus, this study identifies PWWP2A as a complex chromatin-binding protein that serves to direct the deacetylase complex M1HR to H2A.Z-containing chromatin, thereby promoting changes in histone acetylation levels.

Three commercially available artificial bone substitutes with different compositions, hydroxyapatite (HAp; Neobone^®), carbonate apatite (CO₃Ap; Cytrans^®), and β-tricalcium phosphate (β-TCP; Cerasorb^®), were compared with respect to their physical properties and tissue response to bone, using hybrid dogs. Both Neobone^® (HAp) and Cerasorb^® (β-TCP) were porous, whereas Cytrans^® (CO₃Ap) was dense. Crystallite size and specific surface area (SSA) of Neobone^® (HAp), Cytrans^® (CO₃Ap), and Cerasorb^® (β-TCP) were 75.4 ± 0.9 nm, 30.8 ± 0.8 nm, and 78.5 ± 7.5 nm, and 0.06 m²/g, 18.2 m²/g, and 1.0 m²/g, respectively. These values are consistent with the fact that both Neobone^® (HAp) and Cerasorb^® (β-TCP) are sintered ceramics, whereas Cytrans^® (CO₃Ap) is fabricated in aqueous solution. Dissolution in pH 5.3 solution mimicking Howship's lacunae was fastest in CO₃Ap (Cytrans^®), whereas dissolution in pH 7.3 physiological solution was fastest in β-TCP (Cerasorb^®). These results indicated that CO₃Ap is stable under physiological conditions and is resorbed at Howship's lacunae. Histological evaluation using hybrid dog mandible bone defect model revealed that new bone was formed from existing bone to the center of the bone defect when reconstructed with CO₃Ap (Cytrans^®) at week 4. The amount of bone increased at week 12, and resorption of the CO₃Ap (Cytrans^®) was confirmed. β-TCP (Cerasorb^®) showed limited bone formation at week 4. However, a larger amount of bone was observed at week 12. Among these three bone substitutes, CO₃Ap (Cytrans^®) demonstrated the highest level of new bone formation. These results indicate the possibility that bone substitutes with compositions similar to that of bone may have properties similar to those of bone.

Cerebrovascular and neurodegenerative disorders affect one billion people around the world and result from a combination of genomic, epigenomic, metabolic, and environmental factors. Diagnosis at late stages of disease progression, limited knowledge of gene biomarkers and molecular mechanisms of the pathology, and conventional compounds based on symptomatic rather than mechanistic features, determine the lack of success of current treatments, including current FDA-approved conventional drugs. The epigenetic approach opens new avenues for the detection of early presymptomatic pathological events that would allow the implementation of novel strategies in order to stop or delay the pathological process. The reversibility and potential restoring of epigenetic aberrations along with their potential use as targets for pharmacological and dietary interventions sited the use of epidrugs as potential novel candidates for successful treatments of multifactorial disorders involving neurodegeneration. This manuscript includes a description of the most relevant epigenetic mechanisms involved in the most prevalent neurodegenerative disorders worldwide, as well as the main potential epigenetic-based compounds under investigation for treatment of those disorders and their limitations.

Vision is the sense humans rely on most to navigate the world and survive. A tremendous amount of research has focused on understanding the neural circuits for vision and the developmental mechanisms that establish them. The eye-to-brain, or 'retinofugal' pathway remains a particularly important model in these contexts because it is essential for sight, its overt anatomical features relate to distinct functional attributes and those features develop in a tractable sequence. Much progress has been made in understanding the growth of retinal axons out of the eye, their selection of targets in the brain, the development of laminar and cell type-specific connectivity within those targets, and also dendritic connectivity within the retina itself. Moreover, because the retinofugal pathway is prone to degeneration in many common blinding diseases, understanding the cellular and molecular mechanisms that establish connectivity early in life stands to provide valuable insights into approaches that re-wire this pathway after damage or loss. Here we review recent progress in understanding the development of retinofugal pathways and how this information is important for improving visual circuit regeneration.

<h4>Background</h4>In the past decades, substantial effort has been made to explore the genetic influence on brain structural/functional abnormalities in schizophrenia, as well as cognitive impairments. In this work, we aimed to extend previous studies to explore the internal mediation pathway among genetic factor, brain features and cognitive scores in a large Chinese dataset.<h4>Methods</h4>Gray matter (GM) volume, fractional amplitude of low-frequency fluctuations (fALFF), and 4522 schizophrenia-susceptible single nucleotide polymorphisms (SNP) from 905 Chinese subjects were jointly analyzed, to investigate the multimodal association. Based on the identified imaging-genetic pattern, correlations with cognition and mediation analysis were then conducted to reveal the potential mediation pathways.<h4>Findings</h4>One linked imaging-genetic pattern was identified to be group discriminative, which was also associated with working memory performance. Particularly, GM reduction in thalamus, putamen and bilateral temporal gyrus in schizophrenia was associated with fALFF decrease in medial prefrontal cortex, both were also associated with genetic factors enriched in neuron development, synapse organization and axon pathways, highlighting genes including CSMD1, CNTNAP2, DCC, GABBR2 etc. This linked pattern was also replicated in an independent cohort (166 subjects), which although showed certain age and clinical differences with the discovery cohort. A further mediation analysis suggested that GM alterations significantly mediated the association from SNP to fALFF, while fALFF mediated the association from SNP and GM to working memory performance.<h4>Interpretation</h4>This study has not only verified the impaired imaging-genetic association in schizophrenia, but also initially revealed a potential genetic-brain-cognition mediation pathway, indicating that polygenic risk factors could exert impact on phenotypic measures from brain structure to function, thus could further affect cognition in schizophrenia.

Mitochondria are dynamic organelles that undergo fusion, fission, movement, and mitophagy. These processes are essential to maintain the normal mitochondrial morphology, distribution, and function. Mitochondrial fusion allows the exchange of intramitochondrial material, whereas the fission process is required to replicate the mitochondria during cell division, facilitate the transport and distribution of mitochondria, and allow the isolation of damaged organelles. Mitochondrial mobility is essential for mitochondrial distribution depending on the cellular metabolic demands. Mitophagy is needed for the elimination of dysfunctional and damaged mitochondria to maintain a healthy mitochondrial population. The mitochondrial dynamic processes are mediated by a number of nuclear-encoded proteins that function in mitochondrial transport, fusion, fission, and mitophagy. Disorders of mitochondrial dynamics are caused by pathogenic variants in the genes encoding these proteins. These diseases have a high clinical variability, and range in severity from isolated optic atrophy to lethal encephalopathy. These disorders include defects in mitochondrial fusion (caused by pathogenic variants in MFN2, OPA1, YME1L1, MSTO1, and FBXL4), mitochondrial fission (caused by pathogenic variants in DNM1L and MFF), and mitochondrial autophagy (caused by pathogenic variants in PINK1 and PRKN). In this review, the molecular machinery and biological roles of mitochondrial dynamic processes are discussed. Subsequently, the currently known diseases related to mitochondrial dynamic defects are presented.

Histochemistry has an important, continuing role in the current assessment of hepatic biopsies and resection specimens. The evaluation of connective tissue elements in the liver can be accomplished with such methods as the Masson trichrome, Snook reticulin, Vierhoff van Gieson, orcein, and Victoria blue stains. The results contribute to the diagnosis of acute and chronic hepatitis, submassive necrosis, venous outflow obstruction, steatohepatitis, and cirrhosis. Fat stains done on frozen sections of liver tissue are routinely performed in the evaluation of donor liver allograft biopsies. Iron stains such as Perls' method and the Prussian blue technique contribute to the recognition of hemochromatosis and hemosiderosis. The rhodanine, orcein, and Timm stains for copper are used in the characterization of chronic cholestatic liver disease and Wilson's disease. Labeling of carbohydrate-based moieties in various disorders is accomplished with the digested and undigested periodic acid-Schiff method, and Congo red or crystal violet stains can be employed to detect amyloid deposition. Lastly, evaluations of the thickness of the cell plates and continuity of the reticulin framework, as seen with the Snook reticulin stain, can contribute to the diagnostic separation of benign from malignant hepatocellular neoplasms.

Autism spectrum disorder (ASD) is characterized by social and communicative impairments and increased repetitive behaviors. These symptoms are often comorbid with increased anxiety. Prenatal exposure to valproic acid (VPA), an anti-seizure and mood stabilizer medication, is a major environmental risk factor of ASD. Given the important role of the serotonergic (5-HT) system in anxiety, we examined the impact of prenatal VPA exposure on the function of dorsal raphe nucleus (DRn) 5-HT neurons. We found that male rats prenatally exposed to VPA exhibited increased anxiety-like behaviors revealed by a decreased time spent on the open arms of the elevated plus maze. Prenatal VPA exposed rats also exhibited a stereotypic behavior as indicated by excessive self-grooming in a novel environment. These behavioral phenotypes were associated with increased electrical activity of putative DRn 5-HT neurons recorded <i>in vitro</i>. Examination of underlying mechanisms revealed that prenatal VPA exposure increased excitation/inhibition ratio in synapses onto these neurons. The effect was mainly mediated by enhanced glutamate but not GABA release. We found reduced paired-pulse ratio (PPR) of evoked excitatory postsynaptic currents (EPSCs) and increased frequency but not amplitude of miniature EPSCs in VPA exposed rats. On the other hand, presynaptic GABA release did not change in VPA exposed rats, as the PPR of evoked inhibitory postsynaptic currents was unaltered. Furthermore, the spike-timing-dependent long-term potentiation at the glutamatergic synapses was occluded, indicating glutamatergic synaptic transmission is maximized. Lastly, VPA exposure did not alter the intrinsic membrane properties of DRn 5-HT neurons. Taken together, these results indicate that prenatal VPA exposure profoundly enhances glutamatergic synaptic transmission in the DRn and increases spontaneous firing in DRn 5-HT neurons, which could lead to increased serotonergic tone and underlie the increased anxiety and stereotypy after prenatal VPA exposure.

Cardiovascular defects occur in 50% of patients with Turner syndrome (TS). The aim of the study was to estimate the usefulness of cardiac magnetic resonance imaging (CMR) and magnetic resonance angiography (angio-MR) as diagnostics in children and adolescents with TS. Forty-one females with TS, aged 13.9 ± 2.2 years, were studied. CMR was performed in 39 patients and angio-MR in 36. Echocardiography was performed in all patients. The most frequent anomalies diagnosed on CMR and angio-MR were as follows: elongation of the ascending aorta (AA) and aortic arch, present in 16 patients (45.7%), a bicuspid aortic valve (BAV), present in 16 patients (41.0%), and partial anomalous pulmonary venous return (PAPVR), present in six patients (17.1%). Aortic dilatation (<i>Z</i>-score > 2) was mostly seen at the sinotubular junction (STJ) (15 patients; 42.8%), the AA (15 patients; 42.8%), the thoracoabdominal aorta at the level of a diaphragm (15 patients; 42.8%), and the transverse segment (14 patients; 40.0%). An aortic size index (ASI) above 2.0 cm/m² was present in six patients (17.1%) and above 2.5 cm/m² in three patients (8.6%). The left ventricular end-diastolic volume (EDV), end-systolic volume (ESV), and stroke volume (SV) were diminished (<i>Z</i>-score < -2) in 10 (25.6%), 9 (23.1%), and 8 patients (20.5%), respectively. A webbed neck was correlated with the presence of vascular anomalies (<i>p</i> = 0.006). The age and body mass index (BMI) were correlated with the diameter of the aorta. Patients with BAV had a greater aortic diameter at the ascending aorta (AA) segment (<i>p</i> = 0.026) than other patients. ASI was correlated with aortic diameter and descending aortic diameter (AD/DD) ratio (<i>p</i> = 0.002; <i>r</i> = 0.49). There was a significant correlation between the right ventricular (<i>p</i> = 0.002, <i>r</i> = 0.46) and aortic diameters at the STJ segment (<i>p</i> = 0.0047, <i>r</i> = 0.48), as measured by echocardiography and CMR. Magnetic resonance can identify cardiovascular anomalies, dilatation of the aorta, pericardial fluid, and functional impairment of the ventricles not detected by echocardiography. BMI, age, BAV, and elongation of the AA influence aortic dilatation. The ASI and AD/DD ratio are important markers of aortic dilatation. The performed diagnostics did not indicate a negative influence of GH treatment on the cardiovascular system.

Marine sponges of the genus <i>Stelletta</i> are well known as rich sources of diverse and complex biologically relevant natural products, including alkaloids, terpenoids, peptides, lipids, and steroids. Some of these metabolites, with novel structures and promising biological activities, have attracted a lot of attention from chemists seeking to perform their total synthesis in parallel to intensive biological studies towards new drug leads. In this review, we summarized the distribution of the chemically investigated <i>Stelletta</i> sponges, the isolation, synthesis and biological activities of their secondary metabolites, covering the literature from 1982 to early 2018.

Essentials MicroRNAs (miRNAs) regulate the molecular networks controlling biological functions such as hemostasis. We utilized novel methods to analyze miRNA-mediated regulation of the hemostatic system. 52 specific miRNA interactions with 11 key hemostatic associated genes were identified. Functionality and drugability of miRNA-19b-3p against antithrombin were demonstrated in vivo. SUMMARY: Background microRNAs (miRNAs) confer robustness to complex molecular networks regulating biological functions. However, despite the involvement of miRNAs in almost all biological processes, and the importance of the hemostatic system for a multitude of actions in and beyond blood coagulation, the role of miRNAs in hemostasis is poorly defined. Objectives Here we comprehensively illuminate miRNA-mediated regulation of the hemostatic system in an unbiased manner. Methods In contrast to widely applied association studies, we used an integrative screening approach that combines functional aspects of miRNA silencing with biophysical miRNA interaction based on RNA pull-downs (miTRAP) coupled to next-generation sequencing. Results Examination of a panel of 27 hemostasis-associated gene 3'UTRs revealed the majority to possess substantial Dicer-dependent silencing capability, suggesting functional miRNA targeting. miTRAP revealed 150 specific miRNA interactions with 14 3'UTRs, of which 52, involving 40 miRNAs, were functionally confirmed. This includes cooperative miRNA regulation of key hemostatic genes comprising procoagulant (F7, F8, F11, FGA, FGG and KLKB1) and anticoagulant (SERPINA10, PROZ, SERPIND1 and SERPINC1) as well as fibrinolytic (PLG) components. Bioinformatic analysis of miRNA functionality reveals established and potential novel links between the hemostatic system and other pathologies, such as cancer, bone metabolism and renal function. Conclusions Our findings provide, along with an in-vivo proof of concept, deep insights into the network of miRNAs regulating the hemostatic system and present a foundation for biomarker discovery and novel targeted therapeutics for correction of de-regulated hemostasis and associated processes in the future. A repository of the miRNA targetome covering 14 hemostatic components is provided.

<h4>Background</h4>CD62P is a platelet α-granule membrane protein, and P10 is a platelet membrane glycoprotein thrombospondin. To better understand the effects of hemodialysis (HD), we have conducted this study to investigate CD62P and P10 in assessing the efficacy of HD in treating patients with end-stage renal disease (ESRD).<h4>Methods</h4>The case group consisted of 111 patients suffering ESRD treated with regular HD and the control group enrolled 117 healthy subjects. Before and after HD treatment, a series of parameters were observed, based on which, CD62P and P10 levels were detected in the patients in two groups before and after HD therapy. The correlation analysis analyzed the correlations of CD62P and P10 markers with serum creatinine (Scr), blood urea nitrogen (BUN), and subjective score; and logistic regression analysis was performed to reveal factors affecting the efficacy of HD.<h4>Results</h4>BUN, Scr, serum phosphorus, intact parathyroid hormone (iPTH), fibrinogen, and β2-microglobulin (β2-MG) decreased while hemoglobin, albumin, and activated partial thromboplastin time increased in the patients suffering ESRD; patients presented with improvements in subjective symptoms and an increase in dry weight. CD62P and P10 levels were lower in post-treatment patients. CD62P and P10 positively correlated with Scr, BUN and subjective score; post-treatment CD62P and P10 levels, BUN, hemoglobin, albumin, triglyceride, iPTH, β2-MG, and fibrinogen were correlated with the efficacy of HD.<h4>Conclusion</h4>CD62P and P10 might be correlated to the efficacy of HD in treating ESRD, in turn providing predictive markers for assessing the ability of HD in treating ESRD.

<h4>Background</h4>A rare and benign cause of isolated aspartate aminotransferase (AST) increase is due to the presence of macro aspartate aminotransferase (macro-AST). Macro-AST is not usually considered by clinicians, leading to a large number of unnecessary and even invasive tests before diagnosed. Most commonly used laboratory methods for detection of macro-enzymes are precipitation with polyethylene glycol (PEG), ultracentrifugation and gel filtration chromatography (GFC).<h4>Methods</h4>We report a case of an asymptomatic patient with persistent isolated AST elevation. PEG precipitation and an alternative method based on the low stability of this macro-enzyme were performed on the patient serum.<h4>Results</h4>Macro-AST was early detected by the laboratory observing a significant decrease in AST concentrations when the serum is stored at 4ºC.<h4>Conclusions</h4>Macro-AST detection based in this method can be very useful as screening, especially in those laboratories where other technologies are not available. Macro-AST must be considered as cause of unexplained isolated AST elevations before prior investigations to avoid the consequent cost and potential harm to the patient. In our case, early detection of macro-AST with this method avoided unnecessary treatment and invasive test such as liver biopsy.

<h4>Purpose</h4>The molecular events that determine intestinal cell differentiation are poorly understood and it is unclear whether it is primarily a passive event or an active process. It is clinically important to gain a greater understanding of the process, because in colorectal cancer, the degree of differentiation of a tumor is associated with patient survival. <i>SGK1</i> has previously been identified as a gene that is principally expressed in differentiated intestinal cells. In colorectal cancer, there is marked downregulation of <i>SGK1</i> compared with normal tissue.<b>Experimental Design:</b> An inducible <i>SGK1</i> viral overexpression system was utilized to induce reexpression of <i>SGK1</i> in colorectal cancer cell lines. Transcriptomic and phenotypic analyses of these colorectal cancer lines was performed and validation in mouse and human cohorts was performed.<h4>Results</h4>We demonstrate that <i>SGK1</i> is upregulated in response to, and an important controller of, intestinal cell differentiation. Reexpression of <i>SGK1</i> in colorectal cancer cell lines results in features of differentiation, decreased migration rates, and inhibition of metastasis in an orthotopic xenograft model. These effects may be mediated, in part, by SGK1-induced PKP3 expression and increased degradation of MYC.<h4>Conclusions</h4>Our results suggest that <i>SGK1</i> is an important mediator of differentiation of colorectal cells and may inhibit colorectal cancer metastasis.

Hydroxysteroid 17-β dehydrogenase 13 (HSD17B13) is a lipid droplet-associated protein; its gene-encoding variants affect the chronic liver diseases, including nonalcoholic fatty liver disease (NAFLD). To estimate the effect of rs72613567, a splice variant with an adenine insertion (A-INS), on NAFLD susceptibility and severity, we performed a case-control study with 609 individuals. We investigated the effect of carrying the A-INS allele in 356 patients with biopsy-proven disease and explored the relationship between rs72613567 genotypes and the hepatic transcriptome. The A-INS allele protected against NAFLD [odds ratio (OR) per adenine allele = 0.667; 95% CI, 0.486-0.916; <i>P</i> = 0.012]; this effect was nonsignificant when logistic regression analysis included BMI. The A-INS allele protected against nonalcoholic steatohepatitis (NASH) (OR = 0.612; 95% CI, 0.388-0.964; <i>P</i> = 0.033), ballooning degeneration (OR = 0.474; 95% CI, 0.267-0.842; <i>P</i> = 0.01), lobular inflammation (OR = 0.475; 95% CI, 0.275-0.821; <i>P</i> = 0.007), and fibrosis (OR = 0.590; 95% CI, 0.361-0.965; <i>P</i> = 0.035). In patients carrying A-INS, HSD17B13 levels decreased proportionally to allele dosage. Whole-transcriptome genotype profiling showed overrepresented immune response-related pathways. Thus, the rs72613567 A-INS allele reduces the risk of NASH and progressive liver damage and may become a therapeutic target.

Barrett's oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett's oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett's oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett's oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett's oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett's oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

Atypical psychosis (similar to acute and transient psychotic disorder, brief psychotic disorder) is highly heritable, but the causal genes remain unidentified. We conducted whole-genome sequencing on multiplex Japanese families with atypical psychosis. The patient group of interest shows acute psychotic features including hallucinations, delusions, and catatonic symptoms while they often show good prognosis after the onset. In addition to the next-generation analysis, HLA typing has been conveyed to check the similarity with autoimmune disease, such as systemic lupus erythematosus (SLE). Shared causal polymorphisms in the Deleted in Colorectal Carcinoma, Netrin 1 receptor (DCC) gene were found in one multiplex family with three patients, and variants in the RNA 3'-Terminal Phosphate Cyclase (RTCA) and One Cut Homeobox 2 (ONECUT2) genes were found to be shared in seven patients. Next-generation sequencing analysis of the MHC region (previously suggested to be a hot region in atypical psychosis) using HLA typing (HLA-DRB1) revealed a common vulnerability with SLE (systemic lupus erythematosus) among five patients. This finding demonstrates the shared etiology between psychotic symptoms and autoimmune diseases at the genetic level. Focusing on a specific clinical phenotype is key for elucidating the genetic factors that underlie the complex traits of psychosis.

Impaired hippocampal synaptic plasticity contributes to cognitive impairment in Huntington's disease (HD). However, the molecular basis of such synaptic plasticity defects is not fully understood. Combining live-cell nanoparticle tracking and super-resolution imaging, we show that AMPAR surface diffusion, a key player in synaptic plasticity, is disturbed in various rodent models of HD. We demonstrate that defects in the brain-derived neurotrophic factor (BDNF)-tyrosine receptor kinase B (TrkB) signaling pathway contribute to the deregulated AMPAR trafficking by reducing the interaction between transmembrane AMPA receptor regulatory proteins (TARPs) and the PDZ-domain scaffold protein PSD95. The disturbed AMPAR surface diffusion is rescued by the antidepressant drug tianeptine via the BDNF signaling pathway. Tianeptine also restores the impaired LTP and hippocampus-dependent memory in different HD mouse models. These findings unravel a mechanism underlying hippocampal synaptic and memory dysfunction in HD, and highlight AMPAR surface diffusion as a promising therapeutic target.

Aptamers are short single-stranded DNA or RNA oligonucleotide ligand molecules with a unique three-dimensional shape, capable of binding to a defined molecular target with high affinity and specificity. Since their discovery, aptamers have been developed for various applications, including molecular imaging, particularly nuclear imaging that holds the highest potential for the clinical translation of aptamer-based molecular imaging probes. Their easy laboratory production without any batch-to-batch variations, their high stability, their small size with no immunogenicity and toxicity, and their flexibility to incorporate various functionalities without compromising the target binding affinity and specificity make aptamers an attractive class of targeted-imaging agents. Aptamer technology has been utilized in nuclear medicine imaging techniques, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET), as highly sensitive and accurate biomedical imaging modalities towards clinical diagnostic applications. However, for aptamer-targeted PET and SPECT imaging, conjugation of appropriate radionuclides to aptamers is crucial. This review summarizes various strategies to link the radionuclides to chemically modified aptamers to accomplish aptamer-targeted PET and SPECT imaging.

The objective of this study was to evaluate the changes in the goose embryo transcriptome during feather development. RNA-Sequencing (RNA-Seq) was used to find the transcriptome profiles of feather follicles from three stages of embryonic dorsal skin at embryonic day 13, 18, and 28 (E13, E18, E28). The results showed that 3001, 6634, and 13,780 genes were differently expressed in three stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) in E13 vs. E18 were significantly mapped into the GO term of extracellular structure organization and the pathway of extracellular matrix (ECM)-receptor interaction. In E18 vs. E28, the top significantly mapped into GO term was the single-organism developmental process; the pathway was also the ECM-receptor interaction. DEGs in E13 vs. E28 were significantly mapped into the GO term of the multicellular organismal process and the pathway of cell adhesion molecules. Subsequently, the union of DEGs was categorized by succession cluster into eight profiles, which were then grouped into four ideal profiles. Lastly, the seven genes spatio-temporal expression pattern was confirmed by real-time PCR. Our findings advocate that interleukin 20 receptor subunit alpha (<i>IL20RA</i>), interleukin 6 receptor (<i>IL6R</i>), interleukin 1 receptor type 1 (<i>IL-1R1</i>), Wnt family member 3A (<i>WNT3A</i>), insulin-like growth factor binding protein 3 (<i>IGFBP3</i>), bone morphogenetic protein 7 (<i>BMP7</i>), and secreted-frizzled related protein 2 (<i>SFRP2</i>) might possibly play vital roles in skin and feather follicle development and growth processes.

<h4>Introduction</h4>Increasing evidence suggests a role for the gut microbiome in central nervous system disorders and a specific role for the gut-brain axis in neurodegeneration. Bile acids (BAs), products of cholesterol metabolism and clearance, are produced in the liver and are further metabolized by gut bacteria. They have major regulatory and signaling functions and seem dysregulated in Alzheimer's disease (AD).<h4>Methods</h4>Serum levels of 15 primary and secondary BAs and their conjugated forms were measured in 1464 subjects including 370 cognitively normal older adults, 284 with early mild cognitive impairment, 505 with late mild cognitive impairment, and 305 AD cases enrolled in the AD Neuroimaging Initiative. We assessed associations of BA profiles including selected ratios with diagnosis, cognition, and AD-related genetic variants, adjusting for confounders and multiple testing.<h4>Results</h4>In AD compared to cognitively normal older adults, we observed significantly lower serum concentrations of a primary BA (cholic acid [CA]) and increased levels of the bacterially produced, secondary BA, deoxycholic acid, and its glycine and taurine conjugated forms. An increased ratio of deoxycholic acid:CA, which reflects 7α-dehydroxylation of CA by gut bacteria, strongly associated with cognitive decline, a finding replicated in serum and brain samples in the Rush Religious Orders and Memory and Aging Project. Several genetic variants in immune response-related genes implicated in AD showed associations with BA profiles.<h4>Discussion</h4>We report for the first time an association between altered BA profile, genetic variants implicated in AD, and cognitive changes in disease using a large multicenter study. These findings warrant further investigation of gut dysbiosis and possible role of gut-liver-brain axis in the pathogenesis of AD.

Hepcidin is the major regulator of systemic iron metabolism, while the role of this peptide in the brain has just recently been elucidated. Studies suggest a dual role of hepcidin in neuronal iron load and inflammation. This is important since neuronal iron load and inflammation are pathophysiological processes frequently associated with neurodegeneration. Furthermore, manipulation of hepcidin activity has recently been used to recover neuronal damage due to brain inflammation in animal models and cultured cells. Therefore, understanding the mechanistic insights of hepcidin action in the brain is important to uncover its role in treating neuronal damage in neurodegenerative diseases.

Fresh aqueous extracts (AGEs) and several thioallyl compounds (TACs) from garlic have an important antimicrobial activity that likely involves their interaction with exposed thiol groups at single aminoacids or target proteins. Since these groups are present in <i>Giardia duodenalis</i> trophozoites, in this work we evaluated the anti-giardial activity of AGE and several garlic's TACs. <i>In vitro</i> susceptibility assays showed that AGE affected trophozoite viability initially by a mechanism impairing cell integrity and oxidoreductase activities while diesterase activities were abrogated at higher AGE concentrations. The giardicidal activities of seven TACs were related to the molecular descriptor HOMO (Highest Occupied Molecular Orbital) energy and with their capacity to modify the -SH groups exposed in giardial proteins. Interestingly, the activity of several cysteine proteases in trophozoite lysates was inhibited by representative TACs as well as the cytopathic effect of the virulence factor giardipain-1. Of these, allicin showed the highest anti-giardial activity, the lower HOMO value, the highest thiol-modifying activity and the greatest inhibition of cysteine proteases. Allicin had a cytolytic mechanism in trophozoites with subsequent impairment of diesterase and oxidoreductase activities in a similar way to AGE. In addition, by electron microscopy a marked destruction of plasma membrane and endomembranes was observed in allicin-treated trophozoites while cytoskeletal elements were not affected. In further flow cytometry analyses pro-apoptotic effects of allicin concomitant to partial cell cycle arrest at G2 phase with the absence of oxidative stress were observed. In experimental infections of gerbils, the intragastric administration of AGE or allicin decreased parasite numbers and eliminated trophozoites in experimentally infected animals, respectively. These data suggest a potential use of TACs from garlic against <i>G. duodenalis</i> and in the treatment of giardiasis along with their additional benefits in the host's health.

<h4>Background</h4>Basal cell carcinoma (BCC) is a frequent malignant tumor of skin cancers with high morbidity. The objective of this study was to identify critical genes and pathways related to the carcinogenesis of BCC and gain more insights into the underlying molecular mechanisms of BCC.<h4>Materials and methods</h4>The gene expression profiles of GSE7553 and GSE103439 were downloaded from the Gene Expression Omnibus database with 19 tumors and 6 normal skin tissues. Differentially expressed genes (DEGs) were screened between BCC samples and normal tissues, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Subsequently, protein-protein interaction (PPI) network was constructed for these DEGs, and module analysis was performed.<h4>Results</h4>A total of 313 DEGs were obtained. Among them, 222 genes were upregulated and 91 genes were downregulated. Enrichment analysis indicated that the upregulated genes were significantly enriched in cell cycle and mitosis, while the downregulated genes were mainly associated with unsaturated fatty acid metabolic process and cell differentiation. In addition, <i>TOP2A</i>, <i>CDK1</i>, and <i>CCNB1</i> were identified as the top three hub genes ranked by degrees in the PPI network. Meanwhile, three subnetworks were derived, which indicated that these DEGs were significantly enriched in pathways, including "cell cycle", "extracellular matrix-receptor interaction", "basal cell carcinoma", and "hedgehog signaling pathway".<h4>Conclusions</h4>The novel critical DEGs and pathways identified in this study may serve pivotal roles in the carcinogenesis of BCC and indicate more molecular targets for the treatment of BCC.

<h4>Backgrounds</h4>Cholangiocarcinoma (CCA) is epithelial cell malignancy with very poor prognosis. A lot of patients were diagnosed at advanced stage of CCA and no risk factors were identified. There are limited treatment options available for the management of CCA patients. It is urgent to develop effective targeted therapies for the treatment of CCA. miRNAs are small noncoding RNAs that negatively regulate the target genes. In this study, we investigated the role and mechanism of miR-10a-5p in CCA.<h4>Methods</h4>Human CCA cell lines (CCLP1 and SG-231) were transfected with miR-10a-5p mimic or miR-10a-5p inhibitor. qRT-PCR was performed to detect the miR-10a-5p level. Proliferation, colony formation, and apoptosis were analyzed. Luciferase reporter assay was used to explore the targeting of miR-10a-5p on PTEN. For in vivo tumorigenesis assay, CCLP1 cells with stable knockdown of miR-10a-5p or control CCLP1 cells were injected subcutaneously into the flank of the SCID mice and animals were monitored for tumor growth.<h4>Results</h4>miR-10a-5p expression was significantly upregulated in human CCA cell lines (CCLP1 and SG-231). Inhibition of miR-10a-5p significantly suppressed the proliferation and induced apoptosis in CCLP1 and SG-231. PTEN is a direct target of miR-10a-5p in CCA cells.<h4>Conclusion</h4>Inhibition of miR-10a-5p can decrease CCA cells growth by downregulation of Akt pathway. These results indicate that miR-10a-5p may serve as a potential target for the treatment of CCA and help to develop effective therapeutic strategies.

Protein glycosylation plays a key role in various biological processes and disease-related pathological progression. Mass spectrometry (MS)-based glycoproteomics is a powerful approach that provides a system-wide profiling of the glycoproteome in a high-throughput manner. There have been numerous significant technological advances in this field, including improved glycopeptide enrichment, hybrid fragmentation techniques, emerging specialized software packages, and effective quantitation strategies, as well as more dedicated workflows. With increasingly sophisticated glycoproteomics tools on hand, researchers have extensively adapted this approach to explore different biological systems both in terms of in-depth glycoproteome profiling and comparative glycoproteome analysis. Quantitative glycoproteomics enables researchers to discover novel glycosylation-based biomarkers in various diseases with potential to offer better sensitivity and specificity for disease diagnosis. In this review, we present recent methodological developments in MS-based glycoproteomics and highlight its utility and applications in answering various questions in complex biological systems.

No abstract available.

Rare individuals with hypomorphic inactivating mutations in the Huntington’s Disease (HD) gene ( HTT ), identified by CAG repeat expansion in the eponymous neurodegenerative disorder, exhibit variable abnormalities that imply HTT essential roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in Htt , the murine HTT orthologue (in Hdh neoQ20 , Hdh neoQ50 , Hdh neoQ111 mice), were placed over a null allele ( Hdh ex4/5 ). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate alleles yielded perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt -null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development and proposed polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that the HD gene acts both pre- and post-gastrulation and possibly suggest pleiotropic consequences of HTT -lowering therapeutic strategies. <h4>Author Summary</h4> The HTT gene product mutated in Huntington’s Disease (HD) has essential roles during normal organism development, however, still not fully predictable are the functional consequences of its partial inactivation. Our genetic study provides a comprehensive effects’ description of progressively stronger suppression of Htt gene, the murine HTT counterpart. The most severe Htt reduction leads to embryo lethality, while intermediate Htt dosages yield a variety of developmental abnormalities affecting body size, skin, skeletal and ear formation, and hematopoiesis. Complementing molecular analysis in differentiating cells depleted of a functional Htt gene further elucidates genes’ networks dysregulated during organ development and proposes chromatin regulators and short non-coding RNAs as key molecular mediators. Together these findings demonstrate that the HD gene acts both at early and later stages of development, thus possibly suggesting long-term consequences associated to the newest HD therapeutic strategies aimed at lowering the HTT gene product.

<h4>Background</h4>The liver R2* value is widely used as a measure of liver iron but may be confounded by the presence of hepatic steatosis and other covariates.<h4>Purpose</h4>To identify the most influential covariates for liver R2* values in patients with nonalcoholic fatty liver disease (NAFLD).<h4>Study type</h4>Retrospective analysis of prospectively acquired data.<h4>Population</h4>Baseline data from 204 subjects enrolled in NAFLD/NASH (nonalcoholic steatohepatitis) treatment trials.<h4>Field strength</h4>1.5T and 3T; chemical-shift encoded multiecho gradient echo.<h4>Assessment</h4>Correlation between liver proton density fat fraction and R2*; assessment for demographic, metabolic, laboratory, MRI-derived, and histological covariates of liver R2*.<h4>Statistical tests</h4>Pearson's and Spearman's correlations; univariate analysis; gradient boosting machines (GBM) multivariable machine-learning method.<h4>Results</h4>Hepatic proton density fat fraction (PDFF) was the most strongly correlated covariate for R2* at both 1.5T (r = 0.652, P < 0.0001) and at 3T (r = 0.586, P < 0.0001). In the GBM analysis, hepatic PDFF was the most influential covariate for hepatic R2*, with relative influences (RIs) of 61.3% at 1.5T and 47.5% at 3T; less influential covariates had RIs of up to 11.5% at 1.5T and 16.7% at 3T. Nonhepatocellular iron was weakly associated with R2* at 3T only (RI 6.7%), and hepatocellular iron was not associated with R2* at either field strength.<h4>Data conclusion</h4>Hepatic PDFF is the most influential covariate for R2* at both 1.5T and 3T; nonhepatocellular iron deposition is weakly associated with liver R2* at 3T only.<h4>Level of evidence</h4>4 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:1456-1466.

Recent scientific evidence has suggested that long noncoding RNAs (lncRNAs) play an important part in tumorigenesis as an important member of competing endogenous RNAs (ceRNAs). Hundreds of RNA sequence data and relevant clinic information are freely accessible in The Cancer Genome Atlas (TCGA) datasets. However, the role of cancer-related lncRNAs in papillary thyroid cancer (PTC) is not fully understood yet. In this study, we identified 461 RNA sequencing data from TCGA. Subsequently, 45 lncRNAs, 21 miRNAs, and 78 mRNAs were chosen to construct a ceRNA network of PTC. Then, we analyzed the correlation between these 45 PTC-specific lncRNAs and clinic features and patient outcome. Thirty-seven of these lncRNAs were found to be closely related to age, race, gender, lymph node metastasis, TNM staging system, and patient outcome. Additionally, three of them were linked to PTC patient overall survival. Eventually, we selected eight lncRNAs randomly and performed quantificational real-time polymerase chain reaction (qRT-PCR) in 28 newly diagnosed patients with PTC to verify the reliability of the above results. The results of qRT-PCR are totally in agreement with the bioinformatics analysis. Additionally, it was found that HAND2-AS1 was negatively related to tumor size (P < 0.05). The results were consistent with the bioinformatics analysis in TCGA. Taken together, we identified the differentially expressed lncRNAs and constructed a PTC ceRNA network. The study provides a new perspective and supplement for our understanding of lncRNAs in PTC development and reveals potential diagnostic and prognostic markers in PTC.

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here we present single-particle cryo-electron microscopy structures of LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

<h4>Background & aims</h4>Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth.<h4>Methods</h4>FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3).<h4>Results</h4>In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth.<h4>Conclusions</h4>We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

Fisetin was reported to have an anti-proliferative and apoptotic activity as a novel anti-cancer agent in various cancer cell lines. However, the possible molecular targets for the anti-cancer effect of fisetin in human head and neck cancer (HNCC) have not yet been clarified. In this study, the influence of fisetin on the growth and apoptosis of HNCCs were examined. In HSC3 cells, fisetin treatment reduced the viability and induced apoptosis. Through the results from the screening of the expression profile of apoptosis-related genes, sestrin 2 (SESN2) was functionally involved in fisetin-mediated apoptosis showing the knockdown of SESN2 by siRNA clearly restored fisetin-induced apoptosis. In addition, fisetin reduced the protein expression levels of phospho-mTOR (p-mTOR) and Mcl-1, which are the downstream molecules of SESN2. It also induced PARP cleavage by inducing an increase in the expression levels of SESN2 together with reducing mTOR and Mcl-1 proteins in other three HNCCs (MC3, Ca9.22, and HN22). Taken together, our findings suggest that the anti-cancer effect of fisetin on HNCCs is associated with SESN2/mTOR/Mcl-1 signaling axis.

Oxidation of a protein cysteinyl thiol (Cys-SH) to S-sulfenic acid (Cys-SOH) by a reactive oxygen species (e.g., hydrogen peroxide), which is termed protein S-sulfenylation, is a reversible post-translational modification that plays a crucial role in redox regulation of protein function in various biological processes. Due to its intrinsically labile nature, protein S-sulfenylation cannot be directly detected or analyzed. Chemoselective probing has been the method of choice for analyzing S-sulfenylated proteins either in vitro or in situ, as it allows stabilization and direct detection of this transient oxidative intermediate. However, it remains challenging to globally pinpoint the specific S-sulfenylated cysteine sites on complex proteomes and to quantify their dynamic changes upon oxidative stress. This unit describes how a benzothiazine-based chemoselective probe called BTD and mass spectrometry based chemoproteomics can be used to globally and site-specifically identify and quantify protein S-sulfenylation. © 2018 by John Wiley & Sons, Inc.

<h4>Background</h4>Mathematical modeling is a powerful tool to analyze, and ultimately design biochemical networks. However, the estimation of the parameters that appear in biochemical models is a significant challenge. Parameter estimation typically involves expensive function evaluations and noisy data, making it difficult to quickly obtain optimal solutions. Further, biochemical models often have many local extrema which further complicates parameter estimation. Toward these challenges, we developed Dynamic Optimization with Particle Swarms (DOPS), a novel hybrid meta-heuristic that combined multi-swarm particle swarm optimization with dynamically dimensioned search (DDS). DOPS uses a multi-swarm particle swarm optimization technique to generate candidate solution vectors, the best of which is then greedily updated using dynamically dimensioned search.<h4>Results</h4>We tested DOPS using classic optimization test functions, biochemical benchmark problems and real-world biochemical models. We performed [Formula: see text] = 25 trials with [Formula: see text] = 4000 function evaluations per trial, and compared the performance of DOPS with other commonly used meta-heuristics such as differential evolution (DE), simulated annealing (SA) and dynamically dimensioned search (DDS). On average, DOPS outperformed other common meta-heuristics on the optimization test functions, benchmark problems and a real-world model of the human coagulation cascade.<h4>Conclusions</h4>DOPS is a promising meta-heuristic approach for the estimation of biochemical model parameters in relatively few function evaluations. DOPS source code is available for download under a MIT license at http://www.varnerlab.org .

Expansion of the polyglutamine (polyQ) tract in exon 1 of the huntingtin protein (Httex1) leads to Huntington's disease resulting in fatal neurodegeneration. However, it remains poorly understood how polyQ expansions alter protein structure and cause toxicity. Using CD, EPR, and NMR spectroscopy, we found here that monomeric Httex1 consists of two co-existing structural states whose ratio is determined by polyQ tract length. We observed that short Q-lengths favor a largely random-coil state, whereas long Q-lengths increase the proportion of a predominantly α-helical state. We also note that by following a mobility gradient, Httex1 α-helical conformation is restricted to the N-terminal N17 region and to the N-terminal portion of the adjoining polyQ tract. Structuring in both regions was interdependent and likely stabilized by tertiary contacts. Although little helicity was present in N17 alone, each Gln residue in Httex1 enhanced helix stability by 0.03-0.05 kcal/mol, causing a pronounced preference for the α-helical state at pathological Q-lengths. The Q-length-dependent structuring and rigidification could be mimicked in proteins with shorter Q-lengths by a decrease in temperature, indicating that lower temperatures similarly stabilize N17 and polyQ intramolecular contacts. The more rigid α-helical state of Httex1 with an expanded polyQ tract is expected to alter interactions with cellular proteins and modulate the toxic Httex1 misfolding process. We propose that the polyQ-dependent shift in the structural equilibrium may enable future therapeutic strategies that specifically target Httex1 with toxic Q-lengths.

Bipolar disorder (BD) is a mental disorder characterized by alternating periods of depression and mania. Individuals with BD have higher levels of early mortality than the general population, and a substantial proportion of this is due to increased risk for comorbid diseases. To identify the molecular events that underlie BD and related medical comorbidities, we generated imputed whole-genome sequence data using a population-specific reference panel for an extended multigenerational Old Order Amish pedigree (n = 394), segregating BD and related disorders. First, we investigated all putative disease-causing variants at known Mendelian disease loci present in this pedigree. Second, we performed genomic profiling using polygenic risk scores (PRS) to establish each individual's risk for several complex diseases. We identified a set of Mendelian variants that co-occur in individuals with BD more frequently than their unaffected family members, including the R3527Q mutation in APOB associated with hypercholesterolemia. Using PRS, we demonstrated that BD individuals from this pedigree were enriched for the same common risk alleles for BD as the general population (β = 0.416, p = 6 × 10^-4). Furthermore, we find evidence for a common genetic etiology between BD risk and polygenic risk for clinical autoimmune thyroid disease (p = 1 × 10^-4), diabetes (p = 1 × 10^-3), and lipid traits such as triglyceride levels (p = 3 × 10^-4) in the pedigree. We identify genomic regions that contribute to the differences between BD individuals and unaffected family members by calculating local genetic risk for independent LD blocks. Our findings provide evidence for the extensive genetic pleiotropy that can drive epidemiological findings of comorbidities between diseases and other complex traits.