Biology subjects

Mueller, A.

Publications and source records attributed to Mueller, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Multi-omics data integration reveals molecular mechanisms of carfilzomib resistance in multiple myeloma

Multiple myeloma represents a complex hematological malignancy, characterized by its wide array of genetic and clinical events. The introduction of proteasome inhibitors, such as carfilzomib or bortezomib, into the therapeutic landscape has notably enhanced the quality of life and survival rates for patients suffering from this disease. Nonetheless, a significant obstacle in the long-term efficacy of this treatment is the inevitable development of resistance to PIs, posing a substantial challenge in managing the disease effectively. Our study investigates the molecular mechanisms behind carfilzomib resistance by analyzing multi-omics profiles from four multiple myeloma cell lines: AMO-1, KMS-12-PE, RPMI-8226 and OPM-2, together with their carfilzomib-resistant variants. We uncovered a significant downregulation of metabolic pathways linked to strong mitochondrial dysfunction in resistant cells. Further examination of patient samples identified key genes - ABCB1, RICTOR, PACSIN1, KMT2D, WEE1 and GATM - potentially crucial for resistance, guiding us towards promising carfilzomib combination therapies to circumvent resistance mechanisms. The response profiles of tested compounds have led to the identification of a network of gene interactions in resistant cells. We identified two already approved drugs, benidipine and tacrolimus, as potential partners for combination therapy with carfilzomib to counteract resistance. This discovery enhances the clinical significance of our findings.

cancer biology↗

bioRxiv2023

MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence SummaryMITF promote melanoma survival via increasing ROS tolerance.

cancer biology↗

bioRxiv2023

miRNA-mediated inhibition of an actomyosin network in hippocampal pyramidal neurons restricts sociability in adult male mice

Social deficits are frequently observed in patients suffering from neurodevelopmental disorders, but the molecular mechanisms regulating sociability are still poorly understood. We recently reported that the loss of the microRNA cluster miR-379-410 leads to hypersocial behavior and anxiety in mice. Here, we show that ablating miR-379-410 in excitatory neurons of the postnatal mouse hippocampus recapitulates hypersociability, but not anxiety. At the cellular level, miR-379-410 loss in excitatory neurons leads to increased excitatory synaptic transmission and upregulation of an actomyosin gene network. Re-expression of three cluster miRNAs, as well as pharmacological inhibition of the actomyosin activator ROCK, was sufficient to reinstate normal sociability in miR-379-410 knockout mice. Several actomyosin genes and miR-379-410 family members were reciprocally dysregulated in isogenic human iPSC-derived neurons harboring a deletion present in Williams-Beuren-Syndrome patients, which are characterized by hypersocial behavior. Together, our results unveil a novel microRNA-actomyosin pathway involved in the control of sociability.

neuroscience↗

bioRxiv2023

Dynamic ventral disc contraction is necessary for Giardia attachment and host pathology

Giardia lamblia is a common parasitic protist that infects the small intestine and causes giardiasis, resulting in diarrhea, vomiting, weight loss, and malabsorption. Giardiasis leads to cellular damage, including loss of microvilli, disruption of tight junctions, impaired barrier function, enzyme inhibition, malabsorption, and apoptosis. In the host, motile Giardia trophozoites attach to the duodenal microvilli using a unique microtubule organelle called the ventral disc. Despite early observations of disc-shaped depressions in microvilli after parasite detachment, little is known about disc-mediated attachment mechanisms and there little direct evidence showing that parasite attachment causes cellular damage. However, advancements in in vitro organoid models of infection and genetic tools have opened new possibilities for studying molecular mechanisms of attachment and the impact of attachment on the host. Through high-resolution live imaging and a novel disc mutant, we provide direct evidence for disc contraction during attachment, resolving the long-standing controversy of its existence. Specifically, we identify three types of disc movements that characterize contraction, which in combination result in a decrease in disc diameter and volume. Additionally, we investigate the consequences of attachment and disc contractility using an attachment mutant that has abnormal disc architecture. In a human organoid model, we demonstrate that this mutant has a limited ability to break down the epithelial barrier as compared to wild type. Based on this direct evidence, we propose a model of attachment that incorporates disc contraction to generates the forces required for the observed "grasping" of trophozoites on the host epithelium. Overall, this work highlights the importance of disc contractility in establishing and maintaining parasite attachment, leading to intestinal barrier breakdown.

microbiology↗

bioRxiv2023

The domed architecture of Giardias ventral disc is necessary for attachment and host pathogenesis

After ingestion of dormant cysts, the widespread protozoan parasite Giardia lamblia colonizes the host gastrointestinal tract via direct and reversible attachment using a novel microtubule organelle, the ventral disc. Extracellular attachment to the host allows the parasite to resist peristaltic flow, facilitates colonization and is proposed to cause damage to the microvilli of host enterocytes as well as disrupt host barrier integrity. The 9 {micro}m in diameter ventral disc is defined by a highly complex architecture of unique protein complexes scaffolded onto a spiral microtubule (MT) array of one hundred parallel, uniformly spaced MT polymers that bend approximately one and a quarter turns to form a domed structure. To investigate the role of disc-mediated attachment in causing epithelial cell damage, we used a new approach to rapidly create a stable quadruple knockout of Giardia of an essential ventral disc protein, MBP, using a new method of CRISPR-mediated gene disruption with multiple positive selectable markers. MBP quadruple KO mutant discs lack the characteristic domed architecture and possess a flattened "crescent" or horseshoe-shaped conformation that lacks the overlapping region, with severe defects in the microribbon-crossbridge (MR-CB) complex structure. MBP KO mutants are also unable to resist fluid flow required for attachment to inert surfaces. Importantly, MBP KO mutants have 100% penetrance off positive selection, which is essential for quantification of in vivo impacts of disc and attachment mutants with host cells. Using a new gastrointestinal organoid model of pathogenesis, we found that MBP KO infections had a significantly reduced ability to cause the barrier breakdown characteristic of wild-type infections. Overall, this work provides direct evidence of the role of MBP in creating the domed disc, as well as the first direct evidence that parasite attachment is necessary for host pathology, specifically epithelial barrier breakdown.

microbiology↗

bioRxiv2023

Evaluating Study Design Rigor in Preclinical Cardiovascular Research: A Replication Study

BackgroundMethodological rigor remains a priority in preclinical cardiovascular research to ensure experimental reproducibility and high-quality research. Limited reproducibility diminishes the translation of preclinical discoveries into medical practice. In addition, lack of reproducibility fosters uncertainty in the publics acceptance of reported research results. MethodsWe evaluated the reporting of methodological practices in preclinical cardiovascular research studies published in leading scientific journals by screening articles for the inclusion of the following study design elements (SDEs): considering sex as a biological variable, randomization, blinding, and sample size power estimation. We screened for these SDEs across articles regarding preclinical cardiovascular research studies published between 2011 and 2021. We replicated and extended a study published in 2017 by Ramirez et al. We hypothesized a higher SDE inclusion across preclinical studies over time, that preclinical studies that include human and animal substudies within the same study will exhibit greater SDE inclusion than animal-only preclinical studies, and that a difference exists in SDE usage between large and small animal models. ResultsSDE inclusion was low; with 15.2% of animal-only studies including both sexes as a biological variable, 30.4% including randomization, 32.1% including blinding, and 8.2% including sample size estimation. The incorporation of SDEs did not significantly increase over the ten-year timeframe in the screened articles. Randomization and sample size estimation differed significantly between animal and human substudies (corrected p=1.85e-05 and corrected p=3.81e-07, respectively.) ConclusionsEvidence of methodological rigor varies depending on the study type and model organisms used. From 2011-2021, SDE reporting within preclinical studies has not increased, suggesting more work is needed to foster the inclusion of rigorous study design elements in cardiovascular research.

systems biology↗

bioRxiv2023

OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion

Insulin secretion is the process whereby insulin-containing granules fuse with the plasma membrane of pancreatic {beta}-cells. Exocytosis is preceded by cargo loading and granule biogenesis at the Golgi, followed by maturation and transport of the secretory granules; processes that require modification of both the protein and lipid composition of the granules. Here, we show that insulin-containing secretory granules form physical contacts with the endoplasmic reticulum. The lipid exchange protein OSBP dynamically redistributes to ER-SG contacts in a process regulated by Ca2+ and cytosolic pH, and contributes to cholesterol loading of the granules. This process depends on granular PI(4)P and ER-localized VAPs, and is positively regulated by granular PI4-kinases and negatively regulated by granule-localized Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and siRNA-mediated knockdown of OSBP suppresses glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at ER-granule contact sites is involved in the exocytic process, and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.

cell biology↗

bioRxiv2022

Lidocaine Induces Apoptosis in Head and Neck Squamous Cell Carcinoma Cells Through Activation of Bitter Taste Receptor T2R14

Head and neck squamous cell carcinomas (HNSCCs) have high mortality and significant treatment-related morbidity. It is vital to discover effective, minimally invasive therapies that improve survival and quality of life. Bitter taste receptors (T2Rs) are expressed in HNSCCs and their activation can induce apoptosis. Lidocaine is a local anesthetic used in various clinical applications which can also activate bitter taste receptor 14 (T2R14). Lidocaine may have some anti-tumor effects in other cancers, but the mechanism has been mysterious. Here, we found that lidocaine causes intracellular Ca2+ mobilization through activation of T2R14 in HSNCC cells. T2R14 activation with lidocaine depolarizes the mitochondrial membrane, inhibits cell proliferation, and induces apoptosis. Concomitant mitochondrial Ca2+ influx, ROS production causes T2R14-dependent accumulation of poly-ubiquinated proteins, suggesting inhibition of the proteasome as a novel component of T2R14-induced apoptosis. Lidocaine may have therapeutic potential in HNSCC as a topical gel or intratumor injection, warranting future clinical studies. HIGHLIGHTSO_LILidocaine activates bitter taste receptor 14 (T2R14) to increase intracellular Ca2+ and decrease cAMP in head and neck squamous cell carcinoma (HSNCC) cells C_LIO_LILidocaine decreases cell viability and cell proliferation, depolarizes the mitochondrial membrane, and causes production of reactive oxygen species (ROS) C_LIO_LIT2R14 activation with lidocaine induces apoptosis and inhibits the ubiquitin proteasome system (UPS) C_LIO_LILung squamous cell carcinoma (SCC) cells and HNSCC tumor spheroids undergo apoptosis or structural disbandment, respectively, with lidocaine C_LI

cancer biology↗

bioRxiv2022

Transcriptional and clonal characterization of cytotoxic CD8+ T cells in crescentic glomerulonephritis

Crescentic glomerulonephritis (cGN), most often caused by anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, is an aggressive form of immune-mediated kidney disease and represents an important cause of end-stage renal failure. Although it is known that T cells infiltrate the kidney in cGN, their precise role in autoimmune kidney disease remains to be fully elucidated. By performing single-cell analysis, we identified activated, clonally expanded CD8+ T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Using an experimental model of cGN, we demonstrated that clonally expanded murine CD8+ T cells highly expressed the cytotoxic molecule granzyme B. Moreover, lack of CD8+ T cells or granzyme B resulted in an ameliorated course of cGN. This was associated with reduced cleaved caspase-3 induction in renal tissue cells. Our data indicate that clonally expanded cytotoxic CD8+ T cells have a previously unrecognized pathogenic function in aggravating immune-mediated kidney disease.

immunology↗

bioRxiv2022

Accessibility and Reproducible Research Practices in Cardiovascular Literature

BackgroundSeveral fields have described low reproducibility of scientific research and poor accessibility in research reporting practices. Although previous reports have investigated accessible reporting practices that lead to reproducible research in other fields, to date no study has explored the extent of accessible and reproducible research practices in cardiovascular science literature. MethodsTo study accessibility and reproducibility in cardiovascular research reporting, we screened 400 randomly selected articles published in 2019 in three top cardiovascular science publications: Circulation, the European Heart Journal, and the Journal of the American College of Cardiology (JACC). We screened each paper for accessible and reproducible research practices using a set of accessibility criteria including protocol, materials, data, and analysis script availability, as well as accessibility of the publication itself. We also quantified the consistency of open research practices within and across cardiovascular study types and journal formats. ResultsWe identified that fewer than 2% of cardiovascular research publications provide sufficient resources (materials, methods, data, and analysis scripts) to fully reproduce their studies. After calculating an accessibility score as a measure of the extent to which an articles makes its resources available, we also showed that the level of accessibility varies across study types (p = 2e-16) and across journals (p = 5.9e-13). We further show that there are significant differences in which study types share which resources. We also describe the relationship between accessibility scores and corresponding author nationality. ConclusionAlthough the degree to which reproducible reporting practices are present in publications varies significantly across journals and study types; current cardiovascular science reports frequently do not provide sufficient materials, protocols, data, or analysis information to reproduce a study. In the future, having higher standards of accessibility mandated by either journals or funding bodies will help increase the reproducibility of cardiovascular research. FundingAuthors Gabriel Heckerman, Arely Campos-Melendez, and Chisomaga Ekwueme were supported by an NIH R25 grant from the National Heart Lung and Blood Institute (R25HL147666). Eileen Tzng was supported by an AHA Institutional Training Award fellowship (18UFEL33960207).

scientific communication and education↗

bioRxiv2022

Isolation-by-distance and population-size history inferences from the coho salmon (Oncorhynchus kisutch) genome

Coho salmon (Oncorhynchus kisutch) are a culturally and economically important species that return from multiyear ocean migrations to spawn in rivers that flow to the Northern Pacific Ocean. Southern stocks of coho salmon have significantly declined over the past quarter century, and unfortunately, conservation efforts have not reversed this trend. To assist in stock management and conservation efforts, we generated two chromosome-level genome assemblies and sequenced 24 RNA-seq libraries to better annotate the coho salmon genome assemblies. We also resequenced the genomes of 83 coho salmon across their North American range to identify nucleotide variants, characterize the broad effects of isolation-by-distance using a genome-wide association analysis approach, and understand the demographic histories of these salmon by modeling population size from genome-wide data. We observed that more than 13% of all SNPs were associated with latitude (before multiple test correction), likely an affect of isolation-by-distance. From demographic history modeling, we estimated that the SNP latitudinal gradient likely developed as recently as 8,000 years ago. In addition, we identified four genes each harboring multiple SNPs associated with latitude; all of these SNPs were also predicted to modify the function of the gene. Three of these genes have roles in cell junction maintenance and may be involved in osmoregulation. This signifies that ocean salinity may have been a factor influencing coho salmon recolonization after the last glaciation period - generating the current pattern of variation in these three genes.

genomics↗

bioRxiv2022

T32 training is associated with increased likelihood of obtaining an academic research faculty position: a cross-sectional study

BackgroundThe main mission of NIH-sponsored institutional training programs such as the T32 is to provide strong research and career training for early career scientists, while preparing those individuals to become leaders working to meet the health-related research needs of the nation. One of the main avenues to pursue health-related research is becoming research faculty at an academic institution. It is therefore important to know whether these programs are succeeding in this mission, or, if barriers exist that prevent trainees from pursuing these careers. MethodsOur institution currently trains ~ 2400 post-doctoral scholars per year, approximately 5% of whom are enrolled in one of our 33 T32 programs. In this study, we 1) compare the professional outcomes of T32 trainees with non-T32 trainees at our institution, and 2) survey past and current T32 trainees in a subset of high-performing cardiovascular programs about the barriers and enablers they experienced to pursuing research-oriented careers. ResultsFormer T32 trainees are significantly more likely to attain appointments as primarily research faculty members, compared to other trainees. Trainees report a perceived lack of stability, the paucity of open positions, and the publish or perish competitive mentality of academia as their top reasons for abandoning careers in academia. However, they were still more likely to choose research over clinical careers after participating in a dedicated T32 program. ConclusionsOur results support the conclusion that structured training programs strengthen the pipeline of young scientists pursuing careers in academic research, including those from underrepresented backgrounds. However, T32 postdoctoral researchers are held back from pursuing academic careers by a perceived lack of stability and high competition for faculty positions. FundingThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

scientific communication and education↗

bioRxiv2022

Novel feature selection methods for construction of accurate epigenetic clocks

Epigenetic clocks allow the accurate prediction of age based on the methylation status of specific CpG sites in a variety of tissues. These predictive models can be used to distinguish the biological age of an organism from its chronological age, and are a powerful tool to measure the effectiveness of aging interventions. There is a growing need for methods to efficiently construct epigenetic clocks. The most common approach is to create clocks using elastic net regression modelling of all measured CpG sites, without first identifying specific features or CpGs of interest. The addition of feature selection approaches provides the opportunity to reduce the cost and time of clock development by decreasing the number of CpG sites included in clocks. Here, we apply both classic feature selection methods and novel combinatorial methods to the development of epigenetic clocks. We perform feature selection on the human whole blood methylation dataset of [~]470,000 CpG features published by Hannum and colleagues (2015). We develop clocks to predict age, using a variety of feature selection approaches, and all clocks have R2 correlation scores of greater than 0.73. The most predictive clock uses 35 CpG sites for a R2 correlation score of 0.87. The five most frequent sites across all clocks are also modelled to build a clock with a R2 correlation score of 0.83. These two clocks are validated on two external datasets where they maintain excellent predictive accuracy and outperform Hannum et als model in accuracy of age prediction despite using significantly less CpGs. We also identify the associated gene regulatory regions of these CpG sites, which may be possible targets for future aging studies. These novel feature selection algorithms will lower the number of sites needed to be sequenced to build clocks and allow conventionally expensive aging epigenetic studies to cost a fraction of what it would normally.

bioinformatics↗

bioRxiv2021

Food interactions observed in a pharmacokinetic investigation comparing two marketed cold preparations (BNO1016 and ELOM-080) after administration to male beagle dogs

The cold remedies Sinupret extract (BNO 1016) and Gelomyrtol forte (ELOM-080) represent the two top-selling cold remedies in Germany nowadays. Whereas BNO 1016 is a typical immediate release coated tablet, ELOM-080 is an enteric-coated soft gelatin capsule. The latter formulation, however, is at risk of pharmacokinetic interactions affecting absorption especially in the case of concomitant food intake. In the present pilot study, we investigated the risk of a possible food effect on BNO 1016 in comparison to ELOM-080 in three male beagle dogs. Single doses of BNO 1016 and ELOM-80 at 80 mg/kg and 160 mg/kg, respectively, were administered under fasting and fed conditions according to a 4-period, 4-treatment within-design. Blood sampling took place until up to 30 h p.a. and plasma concentrations of gentiopicroside, verbenalin and from a further marker analyte (BNO 1016 analytes) as well as 1,8-cineole, limonene and perillic acid (ELOM-080 analytes) were determined. Pharmacokinetic parameters focusing on rate and extent of absorption were derived. BNO 1016 analytes demonstrated a homogenous course in all animals in both, the fasted and fed state. Pharmacokinetic characteristics of a typical immediate release drug formulation were observed for all analytes and a food effect could be ruled out. ELOM-080 analytes also showed a homogeneous picture in the fasted state. However, lag-times (tlag) of up to 2 h p.a. with corresponding tmax values of 3 to 4 h were observed, reflecting a longer gastric residence time of the formulation. In the fed state, ELOM-080 showed significant pharmacokinetic characteristics suggesting a clear food effect. A major observation was a double peak phenomenon that could be observed in two out of three dogs. Furthermore, lag-times of some analytes up to 3-4 h and corresponding tmax values of up to 6-8 h occurred. In contrast to BNO 1016, these findings suggest that, as with other enteric-coated formulations, there may be a significant risk for food effects with ELOM-080 also in humans.

pharmacology and toxicology↗

bioRxiv2021

Absolute proteome quantification in the gas-fermenting acetogen Clostridium autoethanogenum

Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model-acetogen Clostridium autoethanogenum grown on three gas mixtures. We detect prioritisation of proteome allocation for C1 fixation and significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are important. Integration of proteomic and metabolic flux data demonstrated that enzymes catalyse high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied with changing enzyme catalytic rates rather than concentrations. Our work serves as a reference dataset and advances systems-level understanding and engineering of acetogens.

systems biology↗

bioRxiv2021

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFN{beta} and TNF, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. ImportanceMen experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. One Sentence SummaryFollowing SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

microbiology↗

bioRxiv2021

Bacterial galactosemia is caused by cytoplasmic interference of an essential cell wall biosynthesis glycosyltransferase

Bacterial galactosemia or "galactose death," triggered by incomplete galactose metabolism, was first discovered in Escherichia coli and Salmonella six decades ago, and later in many other microorganisms, yet the mechanism for the toxicity and subsequent cell death remains unclear. In Bacillus subtilis, galactosemia is manifested by a buildup of uridine-diphosphate-galactose (UDP-Gal) and a strong toxicity phenotype characterized by cell shape abnormality and rapid cell lysis. Here we present evidence that in B. subtilis, the toxicity is due to inhibition of cell wall biosynthesis through interference of the essential glycosyltransferase MurG that carries out lipid II synthesis from lipid I and uridine-diphosphate-N-acetyl-glucosamine (UDP-GlcNAc). Single-molecule imaging reveals real-time inhibition of cell wall biosynthesis and MurG activities in cells exhibiting toxicity. We further show that in vitro, MurG is able to utilize UDP-Gal as a substrate generating a "toxic" lipid II, causing a potential poisoning effect on peptidoglycan crosslinking. Evidence also suggests a similar mechanism in Vibrio cholerae and Staphylococcus aureus. Lastly, a strong synergistic lethality was seen in S. aureus wild-type cells treated with both galactose and sub-lethal doses of cell-wall antibiotics. Our study provides mechanistic explanation of the toxicity associated with bacterial galactosemia and its potential application in antibacterial solutions. SignificanceGalactosemia is a potentially fatal genetic disorder due to incomplete galactose metabolism, found in both eukaryotic and prokaryotic organisms. The molecular mechanisms of galactosemia-associated toxicity remain unclear in all cases. Here we present evidence that in the bacterium Bacillus subtilis, the toxicity is due to interference of an essential glycosyltransferase, MurG, which concerts lipid I to lipid II during peptidoglycan biosynthesis, by a nucleotide sugar derived from galactose metabolism. This interference leads to a halt of cell wall biosynthesis and structural defects causing rapid cell lysis. Our evidence also suggests a similar mechanism in other bacteria such as Staphylococcus aureus and Vibrio cholerae. Our study may help solve the long-time puzzle of bacterial galactosemia first uncovered six decades ago.

microbiology↗