Biology subjects

Flores, A.

Publications and source records attributed to Flores, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Comparison of intracellular and encapsidated viral RNA reveals selective constraints on reovirus recombination

Recombination can generate genetic diversity among RNA viruses; yet, mechanisms governing recombination for segmented, double-stranded RNA viruses remain poorly understood. Reovirus genomes contain ten segments of double-stranded RNA. Previous analyses suggested low rates of reovirus RNA recombination and led to a proposed mechanism of recombination during minus-strand synthesis. These analyses focused on recombination events detected in genomic double-stranded RNA extracted from purified viral particles. In the current study, we looked deeper into the origins of the observed recombination events by assaying total cellular material, which comprises viral genomic material, as well as replication intermediates, mRNAs, and products of recombination that may not be eligible for packaging into virus particles. Intracellular viral RNA, which is almost exclusively plus-strand RNA, exhibited substantially higher recombination frequency than particle-associated double-stranded RNA. The diversity and frequency of identified junctions were more similar among samples of a given type (intracellular or virion-encapsidated) from distinct infections than between distinct sample types from the same infection. Although potential recombination sites are widely distributed across the genome, a more limited set was used in junctions in packaged viral RNA than in intracellular viral RNA. Together, these observations suggest recombination may occur more frequently than previously recognized during reovirus infection, and many such events likely occur during plus-strand synthesis. However, recombination products generated during viral transcription may be selectively incorporated into viral particles.

microbiology↗

bioRxiv2026

The dendritic spine vicinome: characterizing its crowding in the human brain with petavoxel electron microscopy

For more than a century we have pictured the dendritic spine as the postsynaptic point where a single presynaptic bouton delivers its message. That picture is correct but incomplete, since it overlooks other neuronal elements surrounding each spine. Here we characterize that neighborhood for the first time by counting and measuring the proximity of the elements encircling each postsynaptic spine. We call this neighborhood the vicinome: the full set of neuronal and glial elements surrounding a dendritic spine. We used ground-truth three-dimensional meshes from the H01 petavoxel dataset, a reconstruction of the human cerebral cortex. We analyzed 4550 neighboring elements across 322 spines from 17 pyramidal neurons in cortical layers 2 through 5, with each spine classified as apical or basal. Neighbor number followed a depth gradient, falling from 17.2 {+/-} 4.2 elements per spine in layer 2 apical spines to 12.5 {+/-} 3.1 in layer 5 basal spines. A linear mixed model with a random intercept per neuron confirmed a strong effect of layer (p = 4.3 x 10-5) with large effect sizes (Cohen's d up to 1.45) and no effect of polarity. The minimum spine-to-neighbor distance stayed uniform, near 30 nm in every group, and although some depth-related contrasts were significant, all effect sizes were negligible (Cohen's d below 0.21). We conclude that cortical depth, and not pyramidal neuron polarity, was related to the number of neuronal elements neighboring the spine. We suggest that vicinome crowding could offer a measurable observable for comparison across regions, ages, species, and disease.

neuroscience↗

bioRxiv2026

Unravelling genomic and functional traits of two biocontrol and plant growth-promoting Pseudomonas endophytes

Plant growth-promoting bacterial endophytes represent a sustainable strategy for enhancing agricultural productivity while reducing reliance on synthetic fertilizers and pesticides. This study focused on the genomic and functional characterization of two endophytic bacterial strains, R11F and R19M, isolated from bean and maize roots, respectively. Comparative analyses based on 16S rRNA gene sequences, average nucleotide identity (ANI), and genome-to-genome distance calculations (GGDC) classified both isolates as Pseudomonas palleroniana. Comparative genomic analyses revealed highly conserved genomes containing genes associated with plant colonization, phosphate solubilization, stress adaptation, heavy metal resistance, and hydrocarbon degradation. Genome mining further identified 17 and 18 biosynthetic gene clusters (BGCs) in R11F and R19M, respectively, including non-ribosomal peptide synthetases (NRPS), pyoverdine, NRP-metallophores, RiPP-like compounds, arylpolyenes, {beta}-lactones, terpenes, NAGGN, and hydrogen cyanide. Strain-specific BGCs associated with syringomycin and viscosin biosynthesis were identified in R11F, whereas R19M harbored clusters related to asplenin and kolossin biosynthesis. In vitro assays confirmed indole production, phosphate solubilization, and siderophore production, as well as the ability of both strains to grow in nitrogen-free medium. Both strains significantly inhibited the growth of Fusarium oxysporum, Phytophthora cinnamomi, and Colletotrichum gloeosporioides. Furthermore, plant inoculation assays demonstrated host-dependent growth promotion, with R11F showing the most consistent improvements in plant growth parameters in tomato, wheat, and lentil. Overall, the integration of comparative genomics and experimental validation demonstrates that P. palleroniana R11F and R19M possess complementary traits associated with plant growth promotion, pathogen suppression, saline stress adaptation, and bioremediation.

microbiology↗

bioRxiv2026

Rotating a petavoxel reconstruction exposes the viewing-angle bias inherent to Golgi-Cox and confocal dendritic-spine classification

Dendritic spines are the principal postsynaptic sites of excitatory transmission. For over a century, their shape has been sorted into discrete categories such as filopodia, thin, long thin, stubby, mushroom, and branched, largely by Golgi-Cox impregnation and, more recently, confocal microscopy. However, both approaches share a fundamental limitation. The histological sectioning and single-viewpoint imaging that these methods rely on cannot control the orientation of a spine relative to the observer. Because a spine is a three-dimensional object, the projection seen depends on how its parent dendrite lies within the section. Here, using the publicly available H01 petavoxel reconstruction of human temporal cortex imaged by serial-section electron microscopy (EM), we show that spine-shape classification depends strongly on viewing angle. A total of 445 spines on layer 4 basal dendrites of five pyramidal neurons were classified from an initial viewpoint (Angle 1), then reclassified after rotation in Neuroglancer (Angle 2). Only 20.9% kept their category, so chance-corrected agreement was negligible (Cohens kappa = 0.027). These observations provide direct evidence that the rigid Golgi-Cox and confocal taxonomies conflate true spine morphology with the arbitrary angle of view. Our results, therefore, support recasting spine shape as a three-dimensional continuum, measurable in petavoxel reconstructions such as H01 through free rotation in Neuroglancer. Significance statementThe classification of dendritic spines into discrete shape classes underpins a vast literature on synaptic plasticity, development, and disease. Yet it rests on two-dimensional images whose viewing angle is not controlled. By rotating the same human spines in a nanoscale EM reconstruction, this study shows that four out of five spines change category with viewpoint alone. The finding exposes a systematic bias in Golgi-Cox and confocal classifications. It argues that spine morphology should be treated as a measurable three-dimensional continuum rather than a set of fixed labels.

neuroscience↗

bioRxiv2026

Novel Synthetic Promoter Armed Oncolytic Herpes Simplex Virus For Treatment of PAX3-FOXO1 Positive Rhabdomyosarcoma

BackgroundDue to the difficulty in targeting oncogenic transcription factors, there are no targeted treatments available for pediatric patients afflicted with PAX3-FOXO1 fusion positive alveolar rhabdomyosarcoma. Additionally, alveolar rhabdomyosarcomas are immunologically cold tumors inherently resistant to immune checkpoint blockade. Viral vectors present a unique opportunity to address this gap in treatment strategies as their presence causes immune infiltration, and a virus can be engineered to rely on tumor specific transcriptional networks for its own replication. MethodologyWe engineered a synthetic promoter which relies on PAX3-FOXO1 to drive expression of a downstream gene. We then placed the HSV-1 RL1 gene encoding ICP34.5 under this promoter within the G47{Delta} oncolytic herpes simplex virus (oHSV), generating a novel oHSV targeting cells harboring the PAX3-FOXO1 fusion gene, which we name oRP3Fus ("Orpheus;" oncolytic recombinant G47{Delta} targeting PAX3-FOXO1 virus). PAX3-FOXO1 specificity was determined by qRT-PCR, protein expression, and chromatin immunoprecipitation. Antitumor efficacy of oncolytic virus and anti-PD1 antibody was determined using immuno-competent mice harboring syngeneic PAX3-FOXO1 expressing rhabdomyosarcoma tumors. Bulk RNA sequencing was performed on infected and mock infected fusion positive and negative cells for differential gene expression analysis. ResultsoRP3Fus kills PAX3-FOXO1 positive human rhabdomyosarcoma lines, while remaining non-toxic to normal human skeletal muscle cells in vitro. In immunocompetent mouse models, both novel and parental viruses improved tumor control when combined with anti-PD1, while oRP3Fus treatment increased T cell infiltration within tumors. RNA and DNA extraction from tumors demonstrate viral expansion within oRP3Fus treated tumors up to three weeks post treatment. Meanwhile oRP3Fus treated mice displayed decreased amounts of necroptosis-associated gene expression within normal organs as compared to groups treated with parental virus. Bulk RNA sequencing of fusion positive and negative human rhabdomyosarcoma lines demonstrated PAX3-FOXO1 increases expression of genes involved in the TNFa-NF{kappa}B signaling axis, as well as innate and adaptive immune signaling. Additionally, ICP34.5 expression from oRP3Fus reduces RIPK1 activation. ConclusionsICP34.5-armed oHSV (oRP3Fus) combined with anti-PD1 provides effective antitumor activity against PAX3-FOXO1-positive alveolar rhabdomyosarcoma in vivo and should be further explored for fusion oncogene-driven tumors. What is already known on this topic?PAX3-FOXO1 fusion-positive and negative rhabdomyosarcoma (RMS) is highly resistant to immune checkpoint blockade. Previously, a PAX3-FOXO1 driven oncolytic adenovirus showed efficacy in an immuno-compromised fusion-positive RMS model. In fusion-negative, immuno-competent RMS models, oncolytic herpes viruses have been shown to improve survival when combined with anti-PD1 therapy, but combinational efficacy was only observed with sex-mismatched models and was never tested on fusion-positive RMS tumors in immunocompetent mice. What this study adds?We improved the specificity of the synthetic PAX3-FOXO1 targeted promoter. We also show, for the first time, efficacy of oncolytic herpes simplex virus-1 in immunocompetent mice harboring orthotopic PAX3-FOXO1-expressing RMS tumors. Our study also demonstrates improvement in survival efficacy in sex-matched tumors, whereas previous reports did not observe complete responses except when using sex-mismatched tumor models. How this study might effect research, practice, or policy?The synthetic promoter generated here can be used for future gene therapy or targeting efforts to study PAX3-FOXO1 or target PAX3-FOXO1 tumors. We observe anti-PD1 therapy benefits survival when paired with an oHSV, demonstrating oncolytic virus can sensitize PAX3-FOXO1 expressing RMS tumors to immunotherapy.

Cancer Biology↗

bioRxiv2026

Genetic code expansion enables programmable covalent protein design

Covalent chemistry has transformed small-molecule drug discovery, yet analogous strategies for proteins remain largely inaccessible because covalent warheads cannot be readily integrated into biologics. Conventional genetic code expansion requires engineering a dedicated aminoacyl-tRNA synthetase for each new amino acid, rendering broad warhead screening impractical. Here we introduce AminoX, a platform that bypasses this limitation through direct tRNA acylation, enabling site-specific incorporation of chemically diverse non-standard amino acids (nsAAs), including covalent warhead nsAAs compatible with scalable biologic manufacturing and multifunctional nsAAs. Using a pooled mRNA display workflow, we screened more than 2,000 warhead-position combinations in machine learning-designed de novo miniproteins targeting CTLA-4, enabling parallel interrogation of covalent chemistry, linker geometry, and incorporation site. We confirmed covalent engagement on cells together with enhanced functional blockade. Finally, we demonstrate multifunctional nsAAs that combine covalent warheads with fluorogenic reporters for real-time detection of target engagement, as well as dual nsAA incorporation for macrocyclization and fluorescent imaging of covalent binding on cell surfaces. By uniting synthetic biology, chemical biology, generative protein design, and high-throughput functional selection, AminoX compresses covalent protein engineering timelines by orders of magnitude, accelerating the development of next-generation therapeutics, biosensors, and chemical probes.

synthetic biology↗

bioRxiv2026

Fibronectin 1 is required for suture patency and dysregulated across craniosynostosis models in the mouse

The mammalian skull roof is comprised of calvarial bones, connected through fibrous sutures, that protect the brain and allow for growth. Premature suture closure, or craniosynostosis (CS), impedes expansion, impacting 1 in every 2500 newborns. Despite its genetic heterogeneity, with nearly 80 associated genes, CS manifests as a common phenotype, hinting at a convergent etiological mechanism. Recently, we described how graded expression of extracellular matrix protein Fibronectin (FN1) is required for apical expansion of calvaria and coronal suture patency. Dysregulated FN1 expression has been identified in two human CS syndromes, suggesting its potential as a convergent mechanism of CS. Here, we further investigate the cellular basis for the CS phenotype in the Fn1 mutant mouse. Graded expression of FN1 baso-apically in the cranial mesenchyme was variably dysregulated across mouse models of syndromic and non-syndromic CS and accompanied by diminished apical expansion of frontal bone primordia. In parallel, at later developmental stages we find ectopic osteogenic induction of Six2+ patent suture mesenchyme in the Fn1 mutant. These findings pinpoint FN1 as a crucial regulator of suture patency by modulating calvarial growth and driving cell identity, and differentiation, thus providing a potential target for matrix-mediated treatments.

developmental biology↗

bioRxiv2026

Neurons in the Human Substantia Nigra Respond to Cognitive Boundaries and Predict Memory

Segmenting mnemonic episodes from continuous experience is a key aspect of human episodic memory. The brain constantly forms predictions about what will happen next based on previous experience and knowledge, and prediction errors are thought to signal when a new event begins (cognitive boundaries). Dopamine has been closely linked to prediction error signals, yet it remains unknown how human midbrain neurons are modulated by cognitive boundaries and how their responses influence memory. To address these questions, we recorded activity of individual neurons in the human substantia nigra, a critical brain structure for dopamine production and regulation, while participants undergoing deep brain stimulation surgery watched a series of clips embedded with cognitive boundaries and performed a recognition memory task. We found that neural activity in the substantia nigra was robustly modulated by cognitive boundaries during clip viewing. Moreover, a subset of these boundary-responsive neurons also differentiated novel from familiar images during recognition, and their firing rates were indicative of participants memory success. These findings reveal that neurons in the human substantia nigra carry boundary- and novelty-related signals consistent with prediction error mechanisms that influence the encoding and retrieval of episodic memories.

neuroscience↗

bioRxiv2025

Reovirus recombination is highly selective, and its profiles are primarily dictated by viral gene segment identity

Recombination facilitates the generation of defective viral genomes (DVGs), truncated derivatives of the parental genome that require a helper virus to replicate. Recombination mechanisms are poorly understood for viruses with double-stranded RNA genomes. Two strains of the double-stranded RNA virus reovirus differ in the pattern of packaged DVGs during serial passage. To determine whether the polymerase complex or gene segment sequence contributes to these differences, we exchanged polymerase complexes between the two reovirus strains. We identified DVG patterns using RT-PCR and recombination junction profiles using ClickSeq. Reoviruses synthesized DVGs that maintained the 5' and 3' termini and contained large central deletions. The polymerase complex did not detectably affect DVG pattern following reovirus serial passage or viral recombination junction profiles. Instead, recombination junction profiles correlated with the identity of viral RNA gene segments, even in the presence of a non-native polymerase complex or virus background. Reovirus recombination junction start and stop sites often occur in regions of sequence microhomology. While we observed many instances of short stretches of identical nucleotides within a viral gene segment, only a select few positions were incorporated into recombination junctions. Overall, these data suggest that recombination events that can mediate reovirus DVG formation are highly selective, and properties of viral gene segments primarily dictate where recombination occurs. These observations suggest a model for double-stranded RNA virus recombination in which the polymerase pauses RNA synthesis and re-initiates further along the same template at a specific junction stop site that has sequence homology to the junction start site. IMPORTANCEViral infection gives rise to defective viral genomes, which cannot complete a full replication cycle. Recombination facilitates defective viral genome generation but is understudied for RNA viruses with double-stranded genomes. We found that for a double-stranded RNA virus, reovirus, recombination occurs preferentially at specific sites in the genome that correspond with the identity of the gene segment. Recombination tends to initiate and terminate at sites sharing identical sequences. However, even if these short nucleotide sequences appear multiple times in a gene segment, only specific sites are used for recombination. Our results indicate that reovirus recombination is a highly orchestrated event in which individual gene segments contain the characteristics that drive recombination. These findings suggest that RNA properties, such as sequence and structure, drive recombination for double-stranded RNA viruses, likely through reinitiation after re-hybridization of the newly formed RNA product with the same RNA template molecule at a different location.

microbiology↗

bioRxiv2025

Neuropathological hallmarks during the chronic phase of ischemic stroke in mice and humans

BackgroundImprovements in acute stroke treatment, including endovascular thrombectomy and critical care management, have increased survival rates post-stroke. However, stroke remains a leading cause of long-term disability and many survivors have significant neurological and cognitive deficits. Despite this, the chronic neurological sequelae and underlying secondary injury mechanisms induced by ischemic stroke remain understudied. MethodsThis study examined long-term neurobehavioral recovery and neuropathology at 2- and 6- months post-stroke in young (12-week-old) male C57Bl/6 mice after a 60-minute transient middle cerebral artery occlusion (MCAO) or sham surgery. Behavioral testing included the open field test (OFT), novel object recognition test (NORT), fear conditioning (FC), nesting activity, and tail suspension. Post-mortem brain samples from patients with chronic ischemic stroke were also assessed. Immunohistochemistry (IHC) was performed to assess demyelination (MBP), neuronal apoptosis (TUNEL), and A{beta}42 in human brains. Flow cytometric analysis was performed to assess microglial phenotypes, the chronic neuroimmune landscape, and to evaluate senescent-like phenotypes (SA-{beta}Gal and lipofuscin). Transcriptomic profiling was performed using RNA isolated from the ipsilateral hemisphere in stroke mice. ResultsExperimental stroke caused progressive cognitive and motor decline up to 6 months post-MCAO. IHC and flow cytometric analyses revealed a significant increase in TUNEL-positive neurons, cortical and hippocampal gliosis, white matter degradation, senescent cell accumulation, and altered microglial function. IHC analysis of postmortem human brains shows significantly increased levels of microgliosis, senescent cells and amyloid burden. Transcriptomic analysis revealed that pathways involving apoptosis, microglial activation and the complement pathway were chronically upregulated after stroke. ConclusionOur findings demonstrate that ischemic stroke induces a non-resolving microglial response and accelerated inflamm-aging in the brain, evidenced by premature senescence and elevated production of cytokines within the chronic infarct microenvironment. Senescent-like phenotypes and chronic neurodegenerative disease signatures may contribute to the progressive worsening of cognitive function post-stroke. These results suggest that chronic, ongoing neurodegeneration occurs late after stroke, even in younger mice. Mitigating these detrimental changes may offer viable targets for delayed treatment strategies for stroke.

neuroscience↗

bioRxiv2023

Defining metabolic flexibility in hair follicle stem cell induced squamous cell carcinoma

Among the numerous changes associated with the transformation to cancer, cellular metabolism is one of the first discovered and most prominent[1, 2]. However, despite the knowledge that nearly every cancer is associated with the strong upregulation of various metabolic pathways, there has yet to be much clinical progress on the treatment of cancer by targeting a single metabolic enzyme directly[3-6]. We previously showed that inhibition of glycolysis through lactate dehydrogenase (LDHA) deletion in cancer cells of origin had no effect on the initiation or progression of cutaneous squamous cell carcinoma[7], suggesting that these cancers are metabolically flexible enough to produce the necessary metabolites required for sustained growth in the absence of glycolysis. Here we focused on glutaminolysis, another metabolic pathway frequently implicated as important for tumorigenesis in correlative studies. We genetically blocked glutaminolysis through glutaminase (GLS) deletion in cancer cells of origin, and found that this had little effect on tumorigenesis, similar to what we previously showed for blocking glycolysis. Tumors with genetic deletion of glutaminolysis instead upregulated lactate consumption and utilization for the TCA cycle, providing further evidence of metabolic flexibility. We also found that the metabolic flexibility observed upon inhibition of glycolysis or glutaminolysis is due to post-transcriptional changes in the levels of plasma membrane lactate and glutamine transporters. To define the limits of metabolic flexibility in cancer initiating hair follicle stem cells, we genetically blocked both glycolysis and glutaminolysis simultaneously and found that frank carcinoma was not compatible with abrogation of both of these carbon utilization pathways. These data point towards metabolic flexibility mediated by regulation of nutrient consumption, and suggest that treatment of cancer through metabolic manipulation will require multiple interventions on distinct pathways.

cancer biology↗

bioRxiv2023

Gestational diabetes augments group B Streptococcus perinatal infection through disruptions in maternal immunity and the vaginal microbiota

Group B Streptococcus (GBS) is a pervasive perinatal pathogen, yet factors driving GBS dissemination in utero are poorly defined. Gestational diabetes mellitus (GDM), a complication marked by dysregulated immunity and maternal microbial dysbiosis, increases risk for GBS perinatal disease. We interrogated host-pathogen dynamics in a novel murine GDM model of GBS colonization and perinatal transmission. GDM mice had greater GBS in utero dissemination and subsequently worse neonatal outcomes. Dual-RNA sequencing revealed differential GBS adaptation to the GDM reproductive tract, including a putative glycosyltransferase (yfhO), and altered host responses. GDM disruption of immunity included reduced uterine natural killer cell activation, impaired recruitment to placentae, and altered vaginal cytokines. Lastly, we observed distinct vaginal microbial taxa associated with GDM status and GBS invasive disease status. Our translational model of GBS perinatal transmission in GDM hosts recapitulates several clinical aspects and enables discovery of host and bacterial drivers of GBS perinatal disease.

microbiology↗

bioRxiv2023

Similarity index of ex-Gaussian reaction time signatures

In psychology and cognitive neuroscience, reaction time (RT) series and their ex-Gaussian distributions are commonly used to explore the time course of cognitive processes. This study investigated the hypothesis that successive triads of ex-Gaussian mu, sigma, and tau parameters of reaction-time variability across time can be used to construct a geometrical object we termed ex-Gaussian RT-signature, which could help characterize interindividual differences between congruent and incongruent stimuli. To test this hypothesis, we calculated the similarity index of these geometrical objects in young adult participants without detectable neurological disorders. Our findings show that each participant displayed distinct ex-Gaussian RT-signatures in a Cartesian 3D plot, thus exhibiting distinct psychophysical fingerprints. Furthermore, our results revealed that the ex-Gaussian RT-signatures for incongruent stimuli demonstrated a significantly higher similarity index across participants than congruent stimuli (p<0.001; Cohen d=0.4). We suggest that visualizing this psychophysical signature could serve as a valuable tool in characterizing differences in cognitive functioning between individuals, thus providing insights into the development of personalized medicine.

neuroscience↗

bioRxiv2023

Unrecognized Potent Activities of Colistin Against Clinically Important mcr+ Enterobacteriaceae Revealed in Synergy with Host Immunity

Colistin (COL) is a cationic cyclic peptide that disrupts negatively-charged bacterial cell membranes and frequently serves as an antibiotic of last resort to combat multidrug-resistant Gram-negative bacterial infections. Emergence of the horizontally transferable plasmid-borne mobilized colistin resistance (mcr) determinant and its spread to Gram-negative strains harboring extended-spectrum {beta}-lactamase and carbapenemase resistance genes threatens futility of our chemotherapeutic arsenal. COL is widely regarded to have zero activity against mcr+ patients based on standard antimicrobial susceptibility testing (AST) performed in enriched bacteriological growth media; consequently, the drug is withheld from patients with mcr+ infections. However, these standard testing media poorly mimic in vivo physiology and omit host immune factors. Here we report previously unrecognized bactericidal activities of COL against mcr-1+ isolates of Escherichia coli (EC), Klebsiella pneumoniae (KP), and Salmonella enterica (SE) in standard tissue culture media containing the physiological buffer bicarbonate. Moreover, COL promoted serum complement deposition on the mcr-1+ Gram-negative bacterial surface and synergized potently with active human serum in pathogen killing. At COL concentrations readily achievable with standard dosing, the peptide antibiotic killed mcr-1+ EC, KP, and SE in freshly isolated human blood proved effective as monotherapy in a murine model of mcr-1+ EC bacteremia. Our results suggest that COL, currently ignored as a treatment option based on traditional AST, may in fact benefit patients with mcr-1+ Gram negative infections based on evaluations performed in a more physiologic context. These concepts warrant careful consideration in the clinical microbiology laboratory and for future clinical investigation of their merits in high risk patients with limited therapeutic options.

microbiology↗

bioRxiv2022

Histone H1.0 Couples Cellular Mechanical Behaviors to Chromatin Structure

Tuning of genome structure and function is accomplished by chromatin binding proteins, which determine the transcriptome and phenotype of the cell. We sought to investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that the linker histone H1.0, which compacts nucleosomes into higher order chromatin fibers, controls genome organization and cellular stress response. Histone H1.0 has privileged expression in fibroblasts across tissue types in mice and humans, and modulation of its expression is necessary and sufficient to mount a myofibroblast phenotype in these cells. Depletion of histone H1.0 prevents transforming growth factor beta (TGF-{beta})-induced fibroblast contraction, proliferation and migration in a histone H1 isoform-specific manner via inhibition of a transcriptome comprised of extracellular matrix, cytoskeletal and contractile genes. Histone H1.0 is associated with local regulation of gene expression via mechanisms involving chromatin fiber compaction and reprogramming of histone acetylation, rendering the cell stiffer in response to cytokine stimulation. Knockdown of histone H1.0 prevented locus-specific histone H3 lysine 27 acetylation by TGF-{beta} and decreased levels of both HDAC1 and the chromatin reader BRD4, thereby preventing transcription of a fibrotic gene program. Transient depletion of histone H1.0 in vivo decompacts chromatin and prevents fibrosis in cardiac muscle, thereby linking chromatin structure with fibroblast phenotype in response to extracellular stress. Our work identifies an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling cellular force generation, nuclear organization and gene transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/518399v1_ufig1.gif" ALT="Figure 1"> View larger version (103K): org.highwire.dtl.DTLVardef@11a38e1org.highwire.dtl.DTLVardef@8c4ab4org.highwire.dtl.DTLVardef@7c822aorg.highwire.dtl.DTLVardef@45d02b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Genome mining, phylogenetic, and functional analysis of arsenic (As) resistance operons in Bacillus strains, isolated from As-rich hot spring microbial mats

The geothermal zone of Araro, Mexico, is located within the trans-Mexican volcanic belt, an area with numerous arsenic (As)-rich hot springs. In this study, the draft genome sequence of two endemic Bacillus strains (ZAP17 and ZAP62) from Araro microbial mat hot springs was determined, which were able to grow on arsenate (up to 64 mM) and arsenite (up to 32 mM). Phylogenetic analysis based on 16S rRNA and gyrB sequences, as well as genome sequence analysis based on average nucleotide identity (>96%) and digital DNA-DNA hybridization (>70%), indicated that these strains belong to the Bacillus paralicheniformis ZAP17 and Bacillus altitudinis ZAP62. Furthermore, through genome mining, it was identified two arsenic resistance operons, arsRBC, and arsRBCDA in both strains as potential determinants of arsenic (As) resistance. Predicted ArsA (arsenial pump-driving ATPase), ArsB (Arsenical efflux pump protein), ArsC (Arsenate reductase), ArsD (Arsenical efflux pump protein) and ArsR (Metalloregulator/ars operon repressor) proteins, clearly grouped with their respective clades corresponding to other characterized bacterial species, mainly Firmicutes. To further evaluate the functionality of the ars operons in ZAP17 and ZAP62 strains, our results showed that arsRBC and arsRBCDA genes were expressed in the presence of arsenite (III). Finally, the presence of ars operons in the genome of Bacillus species residing in As-rich environments, such as the Araro hot springs, might be a potential mechanism to survive under such harsh conditions, as well as to design sustainable bioremediation strategies.

microbiology↗

bioRxiv2022

Basal amygdala inputs to the medial prefrontal cortex mediate fear memory strengthening

Survival critically depends on the memorization of stimuli recurrently predicting pleasant or aversive experiences. Memory strengthening by additional learning is a critical process allowing the long-term stabilization of learned aversive experience. However, the underlying neuronal circuits and mechanisms are still largely unknown. Using a combination of single unit recordings and optogenetic manipulations, we observed that strengthened fear responses following successive fear learning episodes led to a remapping in the population of basal amygdala (BA) neurons encoding the conditioned stimulus. Moreover, strengthened fear responses were preferentially carried by BA neurons projecting to the medial prefrontal cortex (mPFC). Selective optogenetic inhibition of BA terminals in the mPFC during a second learning episode prevented fear memory strengthening. Together, our findings suggest that fear memory strengthening with additional training critically depends on the selective remapping of a subpopulation of BA neurons projecting to the mPFC.

neuroscience↗

bioRxiv2021

Hematopoietic stem and progenitor cells improve survival from sepsis by boosting immunomodulatory cells

New therapeutic strategies to reduce sepsis-related mortality are urgently needed, as sepsis accounts for 1 in 5 deaths worldwide. Since hematopoietic stem and progenitor cells (HSPCs) are responsible for producing blood and immune cells, including in response to immunological stress, we explored their potential for treating sepsis. In a mouse model of Group A Streptococcus (GAS)-induced sepsis, severe immunological stress was associated with significant depletion of bone marrow HSPCs and mortality within approximately 5-7 days. We hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation and depletion that can be rescued by infusion of donor HSPCs. Indeed, infusion of 10,000 naive HSPCs into GAS-infected mice resulted in rapid myelopoiesis and a 50-60% increase in overall survival. Surprisingly, mice receiving donor HSPCs displayed a similar pathogen load compared to untreated mice. Flow cytometric analysis revealed a significantly increased number of myeloid-derived suppressor cells in HSPC-infused mice, which correlated with reduced inflammatory cytokine levels and restored HSPC levels. These findings suggest that HSPCs play an essential immunomodulatory role that may translate into new therapeutic strategies for sepsis.

immunology↗