Biology subjects

Henry, C.

Publications and source records attributed to Henry, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Spatiotemporal dynamics of single-stranded DNA intermediates in Escherichia coli

Single-stranded DNA gaps form within the E. coli chromosome during replication, repair and recombination. However, information about the extent of ssDNA creation in the genome is limited. To complement a recent whole-genome sequencing study revealing ssDNA gap genomic distribution, size, and frequency, we used fluorescence microscopy to monitor the spatiotemporal dynamics of single-stranded DNA within live E. coli cells. The ssDNA was marked by a functional fluorescent protein fusion of the SSB protein that replaces the wild type SSB. During log-phase growth the SSB fusion produces a mixture of punctate foci and diffuse fluorescence spread throughout the cytosol. Many foci are clustered. Fluorescent markers of DNA polymerase III frequently co-localize with SSB foci, often localizing to the outer edge of the large SSB features. Novel SSB-enriched features form and resolve regularly during normal growth. UV irradiation induces a rapid increase in SSB foci intensity and produces large features composed of multiple partially overlapping foci. The results provide a critical baseline for further exploration of ssDNA generation during DNA metabolism. Alterations in the patterns seen in a mutant lacking RecB function tentatively suggest associations of particular SSB features with the repair of double strand breaks and post-replication gaps.

biophysics↗

bioRxiv2023

Basolateral amygdala circuits supporting negative emotional bias in a mouse model for depression

Negative emotional bias is an essential hallmark of depression reflected by negative shift in hedonic valence assignment to emotional stimuli. Pleasant cues become less attractive and unpleasant ones more aversive. Given the crucial role of amygdala in valence coding, we hypothesize that specific basolateral amygdala (BLA) circuits alterations might support negative emotional bias associated with depressive states. Using a translational assay, we evaluate odor valence assignment in an animal model for depression chronically administered by corticosterone (CORT). We show spontaneous negative bias in depressive-like mice that attribute more negative valences for both attractive and aversive odors, mimicking thus the bias observed in depressed bipolar patients. Combining CTB and rabies-based tracing with ex vivo measurements of neuronal activity and chemogenetics experiments, we find that the CORT treatment reduces BLA-to-nucleus accumbens (NAc) neuronal activity and increases BLA-to-central amygdala activity, circuits respectively known to be involved in positive and negative valence encoding. Alterations in presynaptic connectivity of BLA-projecting neurons accompany these activity shifts. Interestingly, inputs from the paraventricular thalamus nucleus (PVT) towards BLA-to-NAc neurons are reduced in CORT-treated mice. Finally, chemogenetically activating the BLA-to-NAc circuit attenuates the negative bias in CORT-treated mice as well as the depressive-like phenotype, similarly than Fluoxetine antidepressant treatment. Altogether, we demonstrate that depressive states are associated with negative emotional bias both in human and mice. This bias is supported by activity shifts of specific BLA circuits along with durable presynaptic connectivity changes, but it could be alleviated by antidepressant drug or activity manipulation of altered BLA circuit.

neuroscience↗

bioRxiv2022

Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains

With the success of mRNA vaccines against coronavirus disease 2019 (COVID-19), strategies can now focus on improving vaccine potency, breadth, and stability. We present the design and preclinical evaluation of domain-based mRNA vaccines encoding the wild-type spike-protein receptor-binding (RBD) and/or N-terminal domains (NTD). An NTD-RBD linked candidate vaccine, mRNA-1283, showed improved antigen expression, antibody responses, and stability at refrigerated temperatures (2-8{degrees}C) compared with the clinically available mRNA-1273, which encodes the full-length spike protein. In mice administered mRNA-1283 as a primary series, booster, or variant-specific booster, similar or greater immune responses and protection from viral challenge were observed against wild-type, beta, delta, or omicron (BA. 1) compared with mRNA-1273 immunized mice, especially at lower vaccine dosages. These results support clinical assessment of mRNA-1283 (NCT05137236). One Sentence SummaryA domain-based mRNA vaccine, mRNA-1283, is immunogenic and protective against SARS-CoV-2 and emerging variants in mice.

immunology↗

bioRxiv2022

Inverse correlation between stress response and virulence factor expression in FASII antibiotic-adapted Staphylococcus aureus and consequences for infection

Antibiotics inhibiting the fatty acid synthesis (FASII) pathway of the major pathogen Staphylococcus aureus reach their enzyme targets, but bacteria continue growth by using environmental fatty acids (eFAs) to produce phospholipids. We assessed how extreme changes in membrane phospholipids provoked by FASII-antibiotics affect global S. aureus physiology. Anti-FASII provoked massive lasting expression changes without genomic rearrangements. Several regulators, rather than one master switch, contributed to the timing of anti-FASII adaptation. Numerous virulence and adhesion factors showed decreased levels and/or activity. Conversely, stress response protein levels increased, and correlated with greater tolerance to peroxides. Notably, peroxide priming stimulated eFA incorporation efficiency and facilitated adaptation to FASII inhibition. These findings establish a link between oxidative stress and FA incorporation. Consistent with major shift in protein expression, anti-FASII-adapted S. aureus killed an insect host more slowly but continued multiplying. Thus, while anti-FASII-adapted populations are less equipped to damage the host, they may be better fit for long term survival, and could constitute a reservoir for re-infection.

microbiology↗

bioRxiv2022

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Far fewer connections were found with the PriA/PriC or PriC/Rep pathways, suggesting a primacy role for the PriA/PriB pathway in E. coli. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break (DSB) accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in {Delta}priB cells. Taken together, our results highlight roles for several genes in DSB homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli. Author SummaryAll organisms rely on DNA replication to grow, develop, and reproduce. In bacteria, the cellular machinery that carries out DNA replication is estimated to fail and prematurely dissociate from the genome at least once per cell cycle. As a result, bacteria have evolved "DNA replication restart" mechanisms that resuscitate failed replication reactions. To probe the function and context of DNA replication restart in the bacterium Escherichia coli, we employed a genetic screen to identify genes that were conditionally important in mutant E. coli strains compromised in their ability to perform DNA replication restart. Identification of genes with previously known relationships with DNA replication restart confirmed the robustness of our screen, while additional findings implicated novel genetic relationships. Targeted experiments validated the importance of these genes and provided an explanation for their significance in preventing double-strand DNA breaks in cells, a severe form of DNA damage. Our results help to define specific roles for the genes identified by our screen and elucidate the contextual environment of DNA repair upstream of DNA replication restart in E. coli.

genetics↗

bioRxiv2022

Periplasmic oxidized-protein repair during copper stress in E. coli: a focus on the metallochaperone CusF.

Methionine residues are particularly sensitive to oxidation by reactive oxygen or chlorine species (ROS/RCS), leading to the appearance of methionine sulfoxide in proteins. This post-translational oxidation can be reversed by omnipresent protein repair pathways involving methionine sulfoxide reductases (Msr). In the periplasm of Escherichia coli, the enzymatic system MsrPQ, whose expression is triggered by the RCS, controls the redox status of methionine residues. Here we report that MsrPQ synthesis is also induced by copper stress via the CusSR two-component system, and that MsrPQ plays a role in copper homeostasis by maintaining the activity of the copper efflux pump, CusCFBA. Genetic and biochemical evidence suggest the metallochaperone CusF is the substrate of MsrPQ and our study reveal that CusF methionines are redox sensitive and can be restored by MsrPQ. Thus, the evolution of a CusSR-dependent synthesis of MsrPQ allows keeping copper homeostasis under aerobic conditions by maintenance of the reduced state of Met residues in copper-trafficking proteins. AUTHOR SUMMARYOur study investigates the interconnection between the copper stress response and the methionine redox homeostasis in the Gram-negative bacterium Escherichia coli. We report that the copper-activation of the CusSR two-component system induces the expression of the periplasmic oxidized-protein repair system, MsrPQ. We demonstrate that MsrPQ is crucial for CusCFBA copper efflux pump activity under aerobic conditions as it maintains the periplasmic component CusF in its functional reduced form. Methionine emerges as a critical residue in copper trafficking proteins: however this naturally-selected advantage must be balanced by methionines high susceptibility to oxidation. Therefore the induction of MsrPQ by copper allows copper homeostasis under aerobic conditions, illustrating that E. coli has developed an integrated and dynamic circuit for sensing and counteracting stress caused by copper and oxidants, thus allowing bacteria to adapt to host cellular defences.

microbiology↗

bioRxiv2021

HypVW is an HOCl-Sensing Two Component System in Escherichia coli

Two component systems (TCS) are signalling pathways that allow bacterial cells to sense, respond and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, YedVW has been recently showed to be involved in the regulation of msrPQ, encoding for the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in a YedVW dependant manner, whereas H2O2, NO and paraquat (a superoxide generator) do not. Therefore, YedV appears to be an HOCl-sensing histidine kinase. Based on this finding, we proposed to rename this system HypVW. Moreover, using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HypV (formerly YedV) are important for its activity. Given that HOCl oxidizes preferentially Met residues, we bring evidences that HypV could be activated via the reversible oxidation of its methionine residues, thus conferring to MsrPQ a role in switching HypVW off. Based on these results, we propose that the activation of HypV by HOCl could occur through a Met redox switch. HypVW appears to be the first characterized TCS able to detect HOCl in E. coli. This study represents an important step in understanding the mechanisms of reactive chlorine species resistance in prokaryotes. IMPORTANCEUnderstanding molecularly how bacteria respond to oxidative stress is crucial to fight pathogens. HOCl is one of the most potent industrial and physiological microbiocidal oxidant. Therefore, bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HypVW as an HOCl-sensing two component system in Escherichia coli MG1655 which regulates the expression of the periplasmic HOCl-oxidized proteins repair system MsrPQ. Moreover we bring evidences suggesting that HOCl could activate HypV through a methionine redox switch.

microbiology↗

bioRxiv2021

TranSyT, the Transport Systems Tracker

The importance and rate of development of genome-scale metabolic models have been growing for the last years, increasing the demand for software solutions that automate several steps of this process. However, since TRIAGEs release, software development for automatic integration of transport reactions into models has stalled. Here we present the Transport Systems Tracker (TranSyT), the next iteration of TRIAGE. Unlike its predecessor, TranSyT does not rely on manual curation to expand its internal database, derived from highly-curated records retrieved from the Transporters Classification Database and complemented with information from other data sources. TranSyT compiles information regarding transporters families, transport proteins, and derives reactions into its internal database, making it available for rapid annotation of complete genomes. All transport reactions have GPR associations and can be exported with identifiers from four different metabolite databases. TranSyT is currently available as a plugin for merlin v4.0 and an app for KBase.

bioinformatics↗

bioRxiv2021

Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a global pandemic. Safe and effective COVID-19 vaccines are now available, including mRNA-1273, which has shown 94% efficacy in prevention of symptomatic COVID-19 disease. However, the emergence of SARS-CoV-2 variants has led to concerns of viral escape from vaccine-induced immunity. Several variants have shown decreased susceptibility to neutralization by vaccine-induced immunity, most notably B.1.351 (Beta), although the overall impact on vaccine efficacy remains to be determined. Here, we present the initial evaluation in mice of 2 updated mRNA vaccines designed to target SARS-CoV-2 variants: (1) monovalent mRNA-1273.351 encodes for the spike protein found in B.1.351 and (2) mRNA-1273.211 comprising a 1:1 mix of mRNA-1273 and mRNA-1273.351. Both vaccines were evaluated as a 2-dose primary series in mice; mRNA-1273.351 was also evaluated as a booster dose in animals previously vaccinated with mRNA-1273. The results demonstrated that a primary vaccination series of mRNA-1273.351 was effective at increasing neutralizing antibody titers against B.1.351, while mRNA-1273.211 was effective at providing broad cross-variant neutralization. A third (booster) dose of mRNA-1273.351 significantly increased both wild-type and B.1.351-specific neutralization titers. Both mRNA-1273.351 and mRNA-1273.211 are being evaluated in pre-clinical challenge and clinical studies.

microbiology↗

bioRxiv2021

An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

immunology↗

bioRxiv2021

Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

microbiology↗

bioRxiv2021

Reconstruction of a catalogue of genome-scale metabolic models with enzymatic constraints using GECKO 2.0

Genome-scale metabolic models (GEMs) have been widely used for quantitative exploration of the relation between genotype and phenotype. Streamlined integration of enzyme constraints and proteomics data into GEMs was first enabled by the GECKO method, allowing the study of phenotypes constrained by protein limitations. Here, we upgraded the GECKO toolbox in order to enhance models with enzyme and proteomics constraints for any organism with an available GEM reconstruction. With this, enzyme-constrained models (ecModels) for the budding yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus were generated, aiming to study their long-term adaptation to several stress factors by incorporation of proteomics data. Predictions revealed that upregulation and high saturation of enzymes in amino acid metabolism were found to be common across organisms and conditions, suggesting the relevance of metabolic robustness in contrast to optimal protein utilization as a cellular objective for microbial growth under stress and nutrient-limited conditions. The functionality of GECKO was further developed by the implementation of an automated framework for continuous and version-controlled update of ecModels, which was validated by producing additional high-quality ecModels for Escherichia coli and Homo sapiens. These efforts aim to facilitate the utilization of ecModels in basic science, metabolic engineering and synthetic biology purposes.

systems biology↗

bioRxiv2021

A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

Broadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is "anchored" to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.

immunology↗

bioRxiv2021

mRNA-1273 efficacy in a severe COVID-19 model: attenuated activation of pulmonary immune cells after challenge

The mRNA-1273 vaccine was recently determined to be effective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from interim Phase 3 results. Human studies, however, cannot provide the controlled response to infection and complex immunological insight that are only possible with preclinical studies. Hamsters are the only model that reliably exhibit more severe SARS-CoV-2 disease similar to hospitalized patients, making them pertinent for vaccine evaluation. We demonstrate that prime or prime-boost administration of mRNA-1273 in hamsters elicited robust neutralizing antibodies, ameliorated weight loss, suppressed SARS-CoV-2 replication in the airways, and better protected against disease at the highest prime-boost dose. Unlike in mice and non-human primates, mRNA-1273- mediated immunity was non-sterilizing and coincided with an anamnestic response. Single-cell RNA sequencing of lung tissue permitted high resolution analysis which is not possible in vaccinated humans. mRNA-1273 prevented inflammatory cell infiltration and the reduction of lymphocyte proportions, but enabled antiviral responses conducive to lung homeostasis. Surprisingly, infection triggered transcriptome programs in some types of immune cells from vaccinated hamsters that were shared, albeit attenuated, with mock-vaccinated hamsters. Our results support the use of mRNA-1273 in a two-dose schedule and provides insight into the potential responses within the lungs of vaccinated humans who are exposed to SARS-CoV-2.

immunology↗

bioRxiv2020

Deleterious impacts of inactivity and beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne Muscular Dystrophy

Although it is known that inactivity is deleterious for healthy individuals, less is known about the consequences of inactivity on muscle disease. Reduced activity is frequently encouraged for individuals with congenital muscular dystrophies such as Duchenne Muscular Dystrophy (DMD). We used the zebrafish dmd mutant and a longitudinal design to elucidate the consequences of inactivity versus activity on muscle health. Inactivity worsened muscle structure and survival. We designed four neuromuscular stimulation paradigms loosely based on weight lifting regimens. Each paradigm differentially affected muscle structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES (1) returns gene expression to wild-type levels, (2) increases muscle adhesion to the extracellular matrix (ECM), and (3) remodels the ECM and supports regeneration. Our data indicate that inactivity is deleterious but neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

developmental biology↗

bioRxiv2020

A novel drug-combination screen in zebrafish identifies epigenetic small molecule candidates for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafish dmd model. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure in dmd zebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescued dmd zebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

pharmacology and toxicology↗

bioRxiv2019

Alteration of cardiolipin-dependent mitochondrial coupling in muscle protects against obesity

The tubular shape of mitochondrial cristae depends upon a specific composition of the inner mitochondrial membrane, including cardiolipin that allows strong curvature and promotes optimal organization of ATP synthase. Here we identify Hacd1, which encodes an enzyme involved in very long chain fatty acid biosynthesis, as a key regulator of composition, structure and functional properties of mitochondrial membranes in muscle. In Hacd1-deficient mice, the reduced cardiolipin content was associated with dilation of cristae and caused defective phosphorylating respiration, characterized by absence of proton leak and oxidative stress.\n\nThe skeletal muscle-specific mitochondrial coupling defect produced a global elevation in basal energy expenditure with increased carbohydrate and lipid catabolism, despite decreased muscle mass and locomotor capacities. Mice were protected against diet-induced obesity despite reduced muscle activity, providing an in vivo proof of concept that reducing mitochondrial coupling efficiency in skeletal muscle might be an actionable mechanism in metabolic disease conditions.

cell biology↗

bioRxiv2019

Identification of antibodies targeting the H3N2 hemagglutinin receptor binding site following vaccination of humans

Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly-reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identified several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that were sensitive to substitutions in conventional antigenic sites and were not broadly-reactive. However, we also identified one H3N2 HA RBS-targeting mAb that was exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We determined that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that some HA RBS-targeting antibodies can be surprisingly effective against variable viral strains even if they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.

microbiology↗