The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as 'facilitates chromatin transcription' (FACT1) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair2. However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen-deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3). SPT16 binds nucleosomal DNA and tethers H2A-H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A-H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Although maladaptive decision-making is a defining feature of drug abuse and addiction, we have yet to ascertain how cocaine self-administration disrupts neural signals in anterior cingulate cortex (ACC), a brain region thought to contribute to attentional control. To address this issue, rats were trained on a reward-guided decision-making task; reward value was manipulated by independently varying the size of or the delay to reward over several trial blocks. Subsequently, rats self-administered either a cocaine (experimental group) or sucrose (control) during 12 consecutive days, after which they underwent a 1-month withdrawal period. Upon completion of this period, rats performed the previously learned reward-guided decision-making task while we recorded from single neurons in ACC. We demonstrate that prior cocaine self-administration attenuates attention and attention-related ACC signals in an intake-dependent manner, and that changes in attention are decoupled from ACC firing. These effects likely contribute to the impaired decision-making-typified by chronic substance abuse and relapse-observed after drug use.

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enrichedpatient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients.

Tissue engineering generally utilizes natural or synthetic scaffolds to repair or replace damaged tissues. However, due to the lack of guidance of biological signals, most of the implanted scaffolds have always suffered from poor in vivo cellularization. Herein, we demonstrate a bio-orthogonal reaction-based strategy to realize in situ specific and fast cellularization of tissue engineering scaffold. DBCO-modified PCL-PEG (PCL-PEG-DBCO) polymer was synthesized and then fabricated into PCL-PEG-DBCO film through electrospinning. Meanwhile, azide-labeled macrophages (N3 (+) macrophages) were obtained through metabolic glycoengineering. Through a series of in vitro dynamic and in vivo characterization, DBCO-modified films were noted to dramatically increase the selective capture efficiency and survival rate of N3 (+) cells. Additionally, there is negligible influence of covalent conjugation on cell viability and proliferation, indicating the feasibility of the bio-orthogonal click reaction-based tissue engineering strategy. Overall, this work shows the advantages of an in situ bio-orthogonal click reaction in realizing highly specific, efficient, and long-lasting scaffold cellularization. We anticipate that this general strategy would be widely applicable and useful in tissue engineering and regenerative medicine in the near future.

Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting humans have occurred since the identification of this virus in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozen spillover infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with equilibrium dissociation constant values ranging from several to hundreds at the micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.IMPORTANCE In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain (RBD) of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the IFNL4 gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable in vitro from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4 This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.IMPORTANCE Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner, as patients with a nonfunctional IFNL4 gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the IFNL4 gene acts in a proviral and anti-inflammatory manner. These surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of interferons (IFNs), i.e., the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its noncanonical transcriptional regulation.

Rotavirus, a diarrheal pathogen spread via fecal-oral transmission, is typically characterized by a winter incidence peak in most countries. Unlike for cholera and other waterborne infections, the role of sanitation and socioeconomic factors on the spatial variation of rotavirus seasonality remains unclear. In the current study, we analyzed their association with rotavirus seasonality, specifically the odds of monsoon cases, across 46 locations from 2001 to 2012 in Dhaka. Drinking water from tube wells, compared to other sources, has a clear protective effect against cases during the monsoon, when flooding and water contamination are more likely. This finding supports a significant environmental component of transmission.

Exposure to environmental pollution has been consistently linked to diabetes incidence in humans, but the potential causative mechanisms remain unclear. Given the critical role of regulated insulin secretion in maintaining glucose homeostasis, environmental chemicals that reach the endocrine pancreas and cause beta cell injury are of particular concern. We propose that cytochrome P450 (CYP) enzymes, which are involved in metabolising xenobiotics, could serve as a useful biomarker for direct exposure of islets to pollutants. Moreover, functional CYP enzymes in islets could also impact beta cell physiology. The aim of this study was to determine whether CYP1A enzymes are activated in islets following direct or systemic exposure to environmental pollutants.

This study aimed to investigate the gut microbiome composition in pregnant women with digestive diseases to analyze the relationships between the microflora changes and digestive diseases during pregnancy.

Whole-body computed tomography (WBCT) is the standard diagnostic method for evaluating polytrauma patients. When patients are unable to elevate their arms, the arms are placed along the body, which affects the image quality negatively. Aim of this systematic review is to evaluate the influence of below the shoulder arm positions on image quality of WBCT.

The ability to rapidly adjust gait to cope with unexpected mechanical perturbations declines with ageing. Previous studies, however, have not ensured that gait stability pre-perturbation was equivalent across participants or age groups which may have influenced the outcomes. In this study, we investigate if age-related differences in stability following gait perturbations remain when all participants walk with equivalent stability. We also examine if interlimb transfer of gait adaptations are observed in healthy older adults, by examining if adaptation to repeated perturbations of one leg can benefit stability recovery when the other leg is perturbed. During walking at their stability-normalised walking speeds (young: 1.32 ± 0.07 m/s; older: 1.31 ± 0.13 m/s; normalised to an average margin of stability of 0.05 m), 30 young and 28 older healthy adults experienced ten unpredictable treadmill belt accelerations (the first and last applied to the right leg, the others to the left leg). Using kinematic data, we assessed the margins of stability during unperturbed walking and the first eight post-perturbation recovery steps. Older adults required three more steps to recover during the first perturbation to each leg than the young adults. Yet, after repeated perturbations of the left leg, older adults required only one more step to recover. Interestingly, for the untrained right leg, the older adults could regain stability with three fewer steps, indicating interlimb transfer of the improvements. Age differences in reactive gait stability remain even when participants' walk with equivalent stability. Furthermore, we show that healthy older adults can transfer improvements in balance recovery made during repeated perturbations to one limb to their recovery following a perturbation to the untrained limb.

Long noncoding RNAs (lncRNAs) participate in regulating many biological processes. However, their roles in influenza A virus (IAV) pathogenicity are largely unknown. Here, we analyzed the expression profiles of lncRNAs and mRNAs in H3N2-infected cells and mock-infected cells by high-throughput sequencing. The results showed that 6129 lncRNAs and 50,031 mRNA transcripts in A549 cells displayed differential expression after H3N2 infection compared with mock infection. Among the differentially expressed lncRNAs, 4963 were upregulated, and 1166 were downregulated. Functional annotation and enrichment analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases (KEGG) suggested that target genes of the differentially expressed lncRNAs were enriched in some biological processes, such as cellular metabolism and autophagy. The up- or downregulated lncRNAs were selected and further verified by quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription PCR (RT-PCR). To the best of our knowledge, this is the first report of a comparative expression analysis of lncRNAs in A549 cells infected with H3N2. Our results support the need for further analyses of the functions of differentially expressed lncRNAs during H3N2 infection.

Dysregulation of autophagy is associated with the neurodegenerative processes in Alzheimer's disease (AD), yet it remains controversial whether autophagy is a cause or consequence of AD. We have previously expressed the full-length human APP in Drosophila and established a fly AD model that exhibits multiple AD-like symptoms. Here we report that depletion of CHIP effectively palliated APP-induced pathological symptoms, including morphological, behavioral, and cognitive defects. Mechanistically, CHIP is required for APP-induced autophagy dysfunction, which promotes Aβ production via increased expression of BACE and Psn. Our findings suggest that aberrant autophagy is not only a consequence of abnormal APP activity, but also contributes to dysregulated APP metabolism and subsequent AD pathogenesis.

The effect of steady-state supratherapeutic sertraline (Zoloft) on QT interval was assessed in a single-center, randomized, 3-way crossover, double-blind, placebo- and moxifloxacin-controlled thorough QT study. Healthy adults received sertraline 400 mg/day, moxifloxacin 400 mg, and placebo, with a washout period (≥14 days) between treatments. A 12-lead electrocardiogram was recorded in triplicate before dosing and at selected time points up to 72 hours after dosing. Analysis of covariance using a mixed-effect model with sequence, period, treatment, time, and treatment-by-time interaction as fixed effects; subject within sequence as a random effect; and baseline QT corrected for heart rate using Fridericia formula (QTcF) as a covariate was conducted. A 90% confidence interval for the least squares (LS) mean difference in QTcF between active treatment and placebo was computed for each postdose time point. Exposure-response was assessed using linear mixed-effect modeling. Fifty-four subjects were enrolled. Over 24 hours after dosing, the LS mean difference in QTcF for sertraline versus placebo ranged from 5.597 milliseconds to 9.651 milliseconds. The upper bound of the 90% confidence interval for the LS mean difference exceeded a predefined 10-millisecond significance threshold at the 4-hour postdose time point only (LS mean, 9.651 milliseconds [90% confidence interval, 7.635-11.666]). In the exposure-response analysis, QTcF values increased significantly with increasing sertraline concentration (slope = 0.036 milliseconds/ng/mL; P < .0001). Predicted change from baseline in QTcF at therapeutic maximum plasma sertraline concentration was 3.57 milliseconds. This thorough QTc study demonstrated a positive signal for QTc prolongation for sertraline at the steady-state 400-mg/day dose.

Procrastination is a widespread habit that has been understudied in the realm of health behaviors, especially sleep. This study aimed to examine the cross-sectional relationships between procrastination and multiple dimensions of sleep in a large national sample of US adolescents and young adults. A random sample of 8742 students from 11 US universities provided self-reports of procrastination (measured by the General Procrastination Scale-Short Form with scores ranging from 1 to 5) and sleep behaviors including social jetlag (the absolute difference between mid-sleep times on weeknights and weekend nights), sleep duration (mean weekly, weeknight, and weekend night), insomnia symptoms (trouble falling/staying asleep), daytime sleepiness, and sleep medication use. Multiple linear regression and Poisson regression models adjusted for socio-demographic and academic characteristics as well as response propensity weights. Higher levels of procrastination were significantly associated with greater social jetlag (β = 3.34 min per unit increase in the procrastination score; 95% CI [1.86, 4.81]), shorter mean weekly sleep duration (β = -4.44 min; 95% CI [-6.36, -2.52]), and shorter weeknight sleep duration (β = -6.10 min; 95% CI [-8.37, -3.84]), but not weekend night sleep duration. Moreover, procrastination was associated with insomnia symptoms (Relative Risk (RR) = 1.27; 95% CI [1.19, 1.37]) and daytime sleepiness (RR = 1.32; 95% CI [1.27, 1.38]), but not sleep medication use. The results were robust to adjustment for anxiety and depressive symptoms. Procrastination was associated with greater social jetlag, shorter sleep duration, and worse sleep quality. If causal, the results suggest that interventions to prevent and manage procrastination might help students to improve their sleep health.

Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1-2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts.

Gene expression noise refers to the variation of the expression level of a gene among isogenic cells in the same environment, and has two sources: extrinsic noise arising from the disparity of the cell state and intrinsic noise arising from the stochastic process of gene expression in the same cell state. Due to the low throughput of the existing method for measuring the two noise components, the architectures of intrinsic and extrinsic expression noises remain elusive. Using allele-specific single-cell RNA sequencing, we here estimate the two noise components of 3975 genes in mouse fibroblast cells. Our analyses verify predicted influences of several factors such as the TATA-box and microRNA targeting on intrinsic or extrinsic noises and reveal gene function-associated noise trends implicating the action of natural selection. These findings unravel differential regulations, optimizations, and biological consequences of intrinsic and extrinsic noises and can aid the construction of desired synthetic circuits.

Mitochondria participate in metabolism and signaling. They adapt to the requirements of various cell types. Publicly available expression data permit to study expression dynamics of genes with mitochondrial function (mito-genes) in various cell types, conditions and organisms. Yet, we lack an easy way of extracting these data for mito-genes. Here, we introduce the visual data mining platform mitoXplorer, which integrates expression and mutation data of mito-genes with a manually curated mitochondrial interactome containing ∼1200 genes grouped in 38 mitochondrial processes. User-friendly analysis and visualization tools allow to mine mitochondrial expression dynamics and mutations across various datasets from four model species including human. To test the predictive power of mitoXplorer, we quantify mito-gene expression dynamics in trisomy 21 cells, as mitochondrial defects are frequent in trisomy 21. We uncover remarkable differences in the regulation of the mitochondrial transcriptome and proteome in one of the trisomy 21 cell lines, caused by dysregulation of the mitochondrial ribosome and resulting in severe defects in oxidative phosphorylation. With the newly developed Fiji plugin mitoMorph, we identify mild changes in mitochondrial morphology in trisomy 21. Taken together, mitoXplorer (http://mitoxplorer.ibdm.univ-mrs.fr) is a user-friendly, web-based and freely accessible software, aiding experimental scientists to quantify mitochondrial expression dynamics.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

The histone demethylase LSD1 is a key chromatin regulator that is often deregulated in cancer. Its ortholog, dLsd1 plays a crucial role in Drosophila oogenesis; however, our knowledge of dLsd1 function is insufficient to explain its role in the ovary. Here, we have performed genome-wide analysis of dLsd1 binding in the ovary, and we document that dLsd1 is preferentially associated to the transcription start site of developmental genes. We uncovered an unanticipated interplay between dLsd1 and the GATA transcription factor Serpent and we report an unexpected role for Serpent in oogenesis. Besides, our transcriptomic data show that reducing dLsd1 levels results in ectopic transposable elements (TE) expression correlated with changes in H3K4me2 and H3K9me2 at TE loci. In addition, our results suggest that dLsd1 is required for Piwi dependent TE silencing. Hence, we propose that dLsd1 plays crucial roles in establishing specific gene expression programs and in repressing transposons during oogenesis.