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Background: Insomnia is more prevalent in individuals with Autism Spectrum Disorder (ASD), can worsen core-symptoms and reduces quality of life of both individuals and caregivers. Although ASD is four times more prevalent in males than females, less is known about sex specific sleep differences in autistic individuals. Recent ASD studies suggest that sleep problems may be more severe in females, which aligns with the sex bias seen in insomnia for the general population. We have previously shown that male mice with a mutation in the high confidence ASD gene Shank3, Shank3 ∆C , recapitulate most aspects of the ASD insomnia phenotype. The objective of the present study was to leverage the Shank3 ∆C model to investigate sex-specific effects in sleep using polysomnography., Methods: Adult male and female Shank3 ∆C and wildtype (WT) littermates were first recorded for 24 h of baseline recordings. Subsequently, they were sleep deprived (SD) for five hours via gentle handling and allowed 19 h of recovery sleep to characterize the homeostatic response to SD. Vigilance states (rapid eye movement (REM) sleep, non-rapid eye movement (NREM) sleep and wake) were assigned by manual inspection using SleepSign. Data processing, statistical analysis and visualization were conducted using MATLAB., Results: Sex and genotype effects were found during baseline sleep and after SD. At baseline, male Shank3 ∆C mice sleep less during the dark period (active phase) while female Shank3 ∆C mice sleep less during the light period (rest phase) and sleep more during the dark period. Both male and female Shank3 ∆C mice show reduced spectral power in NREM sleep. We detect a significant effect of sex and genotype in sleep onset latency and homeostatic sleep pressure (sleepiness). In addition, while male Shank3 ∆C mice fail to increase sleep time following SD as seen in WT, female Shank3 ∆C mice decrease sleep time., Conclusions: Overall, our study demonstrates sex differences in sleep architecture and homeostatic response to SD in adult Shank3 ∆C mice. Thus, our study demonstrates an interaction between sex and genotype in Shank3 ∆C mice and supports the use of the Shank3 ∆C model to better understand mechanisms contributing to the sex differences in insomnia in ASD in clinical populations., (© 2024. The Author(s).)

Sleep/wake cycles intricately shape physiological activities including cognitive brain functions, yet the precise molecular orchestrators of sleep remain elusive. Notably, the clinical impact of benzodiazepine drugs underscores the pivotal role of GABAergic neurotransmission in sleep regulation. However, the specific contributions of distinct GABA A receptor subtypes and their principal scaffolding protein, gephyrin, in sleep dynamics remain unclear. The evolving role of synaptic phospho-proteome alterations at excitatory and inhibitory synapses suggests a potential avenue for modulating gephyrin and, consequently, GABA A Rs for sleep through on-demand kinase recruitment. Our study unveils the distinctive roles of two prevalent GABA A receptor subtypes, α1- and α2-GABA A Rs, in influencing sleep duration and electrical sleep activity. Notably, the absence of α1-GABA A Rs emerges as central in sleep regulation, manifesting significant alterations in both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep during dark or active phases, accompanied by altered electroencephalogram (EEG) patterns across various frequencies. Gephyrin proteomics analysis reveals sleep/wake-dependent interactions with a repertoire of known and novel kinases. Crucially, we identify the regulation of gephyrin interaction with ERK1/2, and phosphorylations at serines 268 and 270 are dictated by sleep/wake cycles. Employing AAV-eGFP-gephyrin or its phospho-null variant (S268A/S270A), we disrupt sleep either globally or locally to demonstrate gephyrin phosphorylation as a sleep regulator. In summary, our findings support the local cortical sleep hypothesis and we unveil a molecular mechanism operating at GABAergic synapses, providing critical insights into the intricate regulation of sleep., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Sleep deprivation (SD) has negative effects on brain and body function. Sleep problems are prevalent in a variety of disorders, including neurodevelopmental and psychiatric conditions. Thus, understanding the molecular consequences of SD is of fundamental importance in biology. In this study, we present the first simultaneous bulk and single-nuclear RNA sequencing characterization of the effects of SD in the male mouse frontal cortex. We show that SD predominantly affects glutamatergic neurons, specifically in layers 4 and 5, and produces isoform switching of over 1500 genes, particularly those involved in splicing and RNA binding. At both the global and cell-type specific level, SD has a large repressive effect on transcription, downregulating thousands of genes and transcripts. As a resource we provide extensive characterizations of cell-types, genes, transcripts, and pathways affected by SD. We also provide publicly available tutorials aimed at allowing readers adapt analyses performed in this study to their own datasets., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue., (© The Author(s) 2022. Published by Oxford University Press.)

Glucosyl transferase I (WaaG) in E. coli catalyzes the transfer of an α-d-glucosyl group to the inner core of the lipopolysaccharide (LPS) and plays an important role in the biogenesis of the outer membrane. If its activity could be inhibited, the integrity of the outer membrane would be compromised and the bacterium would be susceptible to antibiotics that are normally prevented from entering the cell. Herein, three libraries of molecules (A, B and C) were docked in the binding pocket of WaaG, utilizing the docking binding affinity as a filter to select fragment-based compounds for further investigations. From the results of the docking procedure, a selection of compounds was investigated by molecular dynamics (MD) simulations to obtain binding free energy (BFE) and K D values for ligands as an evaluation for the binding to WaaG. Derivatives of 1,3-thiazoles ( A7 and A4 ) from library A and 1,3,4-thiadiazole ( B33 ) from library B displayed a promising profile of BFE, with K D < mM, viz., 0.11, 0.62 and 0.04 mM, respectively. Further root-mean-square-deviation (RMSD), electrostatic/van der Waals contribution to the binding and H-bond interactions displayed a favorable profile for ligands A4 and B33 . Mannose and/or heptose-containing disaccharides C1-C4 , representing sub-structures of the inner core of the LPS, were also investigated by MD simulations, and compound C4 2- showed a calculated K is proximate to the glucose-binding site of WaaG. A study of the variation in angle and distance was performed on the different portions of WaaG (N-, the C- domains and the hinge region). The Spearman correlation coefficient between the two variables was close to unity, where both variables increase in the same way, suggesting a conformational rearrangement of the protein during the MD simulation, revealing molecular motions of the enzyme that may be part of the catalytic cycle. Selected compounds were also analyzed by Saturation Transfer Difference (STD) NMR experiments. STD effects were notable for the 1,3-thiazole derivatives D = 0.4 µM. In the presence of UDP-Glc 2- , the best-docked pose of disaccharide C4 2- is proximate to the glucose-binding site of WaaG. A study of the variation in angle and distance was performed on the different portions of WaaG (N-, the C- domains and the hinge region). The Spearman correlation coefficient between the two variables was close to unity, where both variables increase in the same way, suggesting a conformational rearrangement of the protein during the MD simulation, revealing molecular motions of the enzyme that may be part of the catalytic cycle. Selected compounds were also analyzed by Saturation Transfer Difference (STD) NMR experiments. STD effects were notable for the 1,3-thiazole derivatives A4 , A8 and A15 with the apo form of the protein as well as in the presence of UDP for A4 .

The structure of the O-antigen from Escherichia coli reference strain O188 (E. coli O188:H10) has been investigated. The lipopolysaccharide shows a typical nonrandom modal chain-length distribution and the sugar and absolute configuration analysis revealed d-Man, d-Glc, d-GlcN and d-GlcA as major components. The structure of the O-specific polysaccharide was determined using one- and two-dimensional 1 H and 13 C NMR spectroscopy experiments, where inter-residue correlations were identified by 1 H, 13 C-heteronuclear multiple-bond correlation and 1 H, 1 H-NOESY experiments, which revealed that it consists of pentasaccharide repeating units with the following structure: Biosynthetic aspects and NMR analysis are consistent with the presented structure as the biological repeating unit. The O-antigen of Shigella boydii type 16 differs only in that it carries O-acetyl groups to ~50% at O6 of the branch-point mannose residues. A molecular model of the E. coli O188 O-antigen containing 20 repeating units extends ~100 Å, which is similar to the height of the periplasmic portion of polysaccharide co-polymerase Wzz proteins that regulate the O-antigen chain length of lipopolysaccharides in the Wzx/Wzy biosynthetic pathway., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Understanding sleep and its perturbation by environment, mutation, or medication remains a central problem in biomedical research. Its examination in animal models rests on brain state analysis via classification of electroencephalographic (EEG) signatures. Traditionally, these states are classified by trained human experts by visual inspection of raw EEG recordings, which is a laborious task prone to inter-individual variability. Recently, machine learning approaches have been developed to automate this process, but their generalization capabilities are often insufficient, especially across animals from different experimental studies. To address this challenge, we crafted a convolutional neural network-based architecture to produce domain invariant predictions, and furthermore integrated a hidden Markov model to constrain state dynamics based upon known sleep physiology. Our method, which we named SPINDLE (Sleep Phase Identification with Neural networks for Domain-invariant LEearning) was validated using data of four animal cohorts from three independent sleep labs, and achieved average agreement rates of 99%, 98%, 93%, and 97% with scorings from five human experts from different labs, essentially duplicating human capability. It generalized across different genetic mutants, surgery procedures, recording setups and even different species, far exceeding state-of-the-art solutions that we tested in parallel on this task. Moreover, we show that these scored data can be processed for downstream analyzes identical to those from human-scored data, in particular by demonstrating the ability to detect mutation-induced sleep alteration. We provide to the scientific community free usage of SPINDLE and benchmarking datasets as an online server at https://sleeplearning.ethz.ch. Our aim is to catalyze high-throughput and well-standardized experimental studies in order to improve our understanding of sleep., Competing Interests: The authors have declared that no competing interests exist.

The outer membrane of gram-negative bacteria is a permeability barrier that prevents the efficient uptake of molecules with large scaffolds. As a consequence, a number of antibiotic classes are ineffective against gram-negative strains. Herein we carried out a high throughput screen for small molecules that make the outer membrane of Escherichia coli more permeable. We identified MAC13243, an inhibitor of the periplasmic chaperone LolA that traffics lipoproteins from the inner to the outer membrane. We observed that cells were (1) more permeable to the fluorescent probe 1-N-phenylnapthylamine, and (2) more susceptible to large-scaffold antibiotics when sub-inhibitory concentrations of MAC13243 were used. To exclude the possibility that the permeability was caused by an off-target effect, we genetically reconstructed the MAC13243-phenotype by depleting LolA levels using the CRISPRi system.

WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using (14)C-labeled UDP-glucose and LPS purified from a ∆waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC50 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.

How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Factors interacting with core circadian clock components are essential to achieve transcriptional feedback necessary for metazoan clocks. Here, we show that all three members of the Drosophila behavior human splicing (DBHS) family of RNA-binding proteins play a role in the mammalian circadian oscillator, abrogating or altering clock function when overexpressed or depleted in cells. Although these proteins are members of so-called nuclear paraspeckles, depletion of paraspeckles themselves via silencing of the structural noncoding RNA (ncRNA) Neat1 did not affect overall clock function, suggesting that paraspeckles are not required for DBHS-mediated circadian effects. Instead, we show that the proteins bound to circadian promoter DNA in a fashion that required the PERIOD (PER) proteins and potently repressed E-box-mediated transcription but not cytomegalovirus (CMV) promoter-mediated transcription when they were exogenously recruited. Nevertheless, mice with one or both copies of these genes deleted show only small changes in period length or clock gene expression in vivo. Data from transient transfections show that each of these proteins can either repress or activate, depending on the context. Taken together, our data suggest that all of the DBHS family members serve overlapping or redundant roles as transcriptional cofactors at circadian clock-regulated genes.

Purpose: Recurrent cytomegalovirus (CMV) disease is a frequent complication of liver transplantation. Visceral leishmaniosis in a transplant recipient is, on the other hand, extremely rare and only two cases of kala-azar have been described after liver transplantation. Immunosuppressed patients are known to be at risk of Legionella infection and the relationship between infection with this organism and hospital water supplies has been well described. These three diseases carry a high mortality rate. Our report examines the potential relationship between these complications., Clinical Features: We describe the case of a liver transplant recipient who presented the three complications successively and survived. After reviewing the literature, we explore hypotheses linking these infections and discuss treatment strategies., Conclusions: In the patient described, infection with leishmania probably occurred months prior to the clinical presentation, a delay that matches the incubation period of kala-azar. The simultaneous onset of leishmaniosis and of a high CMV viremia may have been a coincidence. However, CMV infection has been shown to be an independent predictor of invasive fungal infection in liver transplant recipients. CMV does indeed have a suppressive effect on the humoral and cellular immune response in vitro as well as in vivo. The clinical manifestations of leishmaniosis may, therefore, have been precipitated in this patient by the additive immunosuppressive effect of antirejection drugs and CMV.

Background: Recurrent infection frequently follows the response to the initial treatment of cytomegalovirus (CMV) infection in solid-organ transplant (SOT) recipients. The objective of this study was to describe the course of CMV infection in SOT patients and to identify factors that would predict protracted CMV infection with recurrences., Methods: Quantitative polymerase chain reaction (PCR) assay for CMV DNA in leukocytes and in plasma were used to assess viral load changes retrospectively in consecutive SOT patients, whose CMV infection episodes had been attended therapeutically or preemptively using quantitative blood culture., Results: Among 101 SOT patients, CMV infection occurred in 63, of whom 32 developed recurrent infection after the initial episode. In patients with recurrent infection, PCR indicated that a majority (27) of recipients had high level of CMV DNA in peripheral blood leukocytes and plasma throughout a protracted (>/=1 month) period including after preemptive or therapeutic ganciclovir courses. Predictors of protracted high-level infection were increasing age, CMV donor seropositivity, and all measures of viral load during the initial episode. CMV recipient seropositivity protected strongly against protracted infection. End of treatment plasma CMV DNA best discriminated between patients who did or did not develop protracted infection., Conclusions: In SOT patients, protracted CMV infection is associated with increasing age, donor seropositivity, recipient seronegativity, and high viral load during the first episode. End of therapy plasma CMV DNA level best predicts the occurrence of protracted infection. In patients with a high risk of protracted infection, prophylaxis is likely to be particularly cost effective.

The incidence of cytomegalovirus (CMV) end-organ disease can be reduced in AIDS patients by oral ganciclovir. However, the cost effectiveness of this prophylaxis is low. Targeting prophylaxis to patients with the highest risk of developing CMV disease might be useful. Several studies have shown the potential of various polymerase chain reaction (PCR) assays and of the antigenemia assay to identify the subset of patients with a definitely higher risk of developing CMV disease. We studied the CMV viral load using quantitative PCR in the leukocytes and plasma (or serum) of 28 patients in the four years before they experienced a CMV event. We observed rising CMV DNA copy numbers in the patients' leukocytes a year before the event. In contrast, plasma or serum copy numbers rose later and in fewer patients. In a control population of 21 profoundly immunodeficient patients (median CD4+ T cell count: 31/mm(3)) without history of CMV disease, only five had detectable CMV DNA in the leukocytes, three of whom had barely above-threshold levels. We suggest that, at the present time, leukocyte CMV DNA PCR might represent a sensitive test providing an early warning signal of increased risk of CMV disease. Performing it twice a year might identify patients at risk so that closer monitoring and targeted prophylaxis can be conducted.