Biology subjects

Hernandez, J.

Publications and source records attributed to Hernandez, J..

30 records · Page 2Linked to original sources

bioRxiv2022

Isolation and characterization of Ovine gammaherpesvirus type 2 from an outbreak of Malignant Catarrhal Fever in Artiodactyla and horses in Mexico.

Ovine gammaherpesvirus 2 (OvHV-2), a member of the Macavirus genus, causes sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease that affects a wide variety of ungulates in addition to horses.This study described an outbreak of SA-MCF that occurred in Mexico and the identification of the OvHV-2 virus through viral isolation and different laboratory techniques such as immunofluorescence (IF), immunoperoxidase (IP), immunohistochemistry (IHC), end point PCR and partial sequencing of the ORF75 gene. The animals involved in this outbreak showed head and eye clinical signs and lesions. Based on the clinical-pathological outcome, buffy coats were taken, and virus isolation was attempted on primary cell cultures of the rabbit testicle. Small clusters of refractile cytomegalic cells characterized the cytopathic effect between 48 and 72 hours postinfection. In addition, inclusion bodies were identified, and cytoplasmic immunoreactivity was observed in the infected cells. The sequences obtained were aligned with OvHV-2 sequences reported in GenBank and revealed a nucleotide identity higher than 98%. The results indicate that the outbreak was caused by OvHV-2 and the horses are susceptible to SA-MCF.

microbiology↗

bioRxiv2022

A cadherin mutation in Celsr3 linked to Tourette Disorder affects dendritic patterning and excitability of cholinergic interneurons

CELSR3 encodes an atypical protocadherin cell adhesion receptor that was recently identified as a high-risk gene for Tourette disorder. A putative damaging de novo variant was inserted into the mouse genome to generate an amino acid substitution within the fifth cadherin repeat. By contrast to Celsr3 constitutive null animals, mice homozygous for the R774H amino acid substitution are viable and have grossly normal forebrain development. The density of cortical and striatal interneuron subpopulations is normal, but 3D geometric analysis of cortical pyramidal neurons and striatal cholinergic interneurons revealed changes to dendritic patterning and types and distributions of spines. Furthermore, patch clamp recordings in cholinergic interneurons located within the sensorimotor striatum uncovered mild intrinsic hyperexcitability. Despite these changes, Celsr3R774H homozygous mice do not show obvious tic-like stereotypies at baseline nor motor learning impairments, but females exhibited perseverative digging behavior. Our findings show that a human mutation in CELSR3 linked to Tourette disorder is sufficient to alter dendritic patterning in the cortex and striatum and also the intrinsic excitability of cholinergic interneurons.

neuroscience↗

bioRxiv2021

A Tissue-Bioengineering Strategy for Modeling Rare Human Kidney Diseases In Vivo

The lack of animal models for certain human diseases precludes our understanding of disease mechanisms and our ability to test new therapies in vivo. Here we generated kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs to recapitulate a rare kidney tumor called angiomylipoma (AML). Organoids derived from TSC2-/- hiPSCs but not from isogenic TSC2+/- or TSC2+/+ hiPSCs shared a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and developed epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be robustly and consistently recapitulated with transgenic mice. Transplantation of multiple TSC2-/- kidney organoids into the kidneys of immunodeficient rats allowed us to recapitulate AML and cystic kidney disease in vivo, in a scalable fashion and with fidelity, and to test the efficiency of rapamycin-loaded nanoparticles as a novel approach to ablate AMLs by inducing apoptosis triggered by mTOR-inhibition. Collectively, these methods represent a novel tissue-bioengineering strategy for rare disease modeling in vivo.

bioengineering↗

bioRxiv2021

Structure and function of one unclassified-class glutathione S-transferase in Leptinotarsa decemlineata

Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation is through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agriculture pest of plants within Solanaceae have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 [A] by x-ray crystallography. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2,4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.

biophysics↗

bioRxiv2021

Inducible Yeast Two-Hybrid with Quantitative Measures

The yeast two-hybrid (Y2H) assay has long been used to identify new protein-protein interaction pairs and to compare relative interaction strengths. Traditional Y2H formats may be limited, however, by use of constitutive strong promoters if expressed proteins have toxic effects or post-transcriptional expression differences in yeast among a comparison group. As a step toward more quantitative Y2H assays, we modified a common vector to use an inducible CUP1 promoter, which showed quantitative induction of several "bait" proteins with increasing copper concentration. Using mouse Nxf1 (homologous to yeast Mex67p) as a model bait, copper titration achieved levels that bracket levels obtained with the constitutive ADH1 promoter. Using a liquid growth assay for an auxotrophic reporter in multiwell plates allowed log-phase growth rate to be used as a measure of interaction strength. These data demonstrate the potential for quantitative comparisons of protein-protein interactions using the Y2H system.

genetics↗

bioRxiv2021

Rhes Deletion Prevents Age-Dependent Selective Motor Deficits and Reduces Phosphorylation of S6K in Huntington Disease Hdh150Q(CAG) Knock-In Mice.

Huntington disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide repeat expansion in the huntingtin (mHTT) protein. This expansion is thought to promote striatal atrophy by a combination of cell- and non-cell-autonomous processes, but the mechanisms are unclear. Previous evidence suggests that the striatal-enriched SUMO E3-like protein Rhes could play a pathological role in HD. Rhes interacts with, and SUMOylates, mHTT and promotes toxicity and Rhes deletion ameliorates the HD phenotype in cell and severe mouse models of HD. However, the effect of Rhes on less severe knock-in models of HD remains obscure. Here, we report that a Hdh(CAG)150 knock-in murine model of HD showed diminished body weight but no changes in locomotor coordination or activity at 80 and 100 weeks of age. Conversely, Rhes deletion did not impact the body weight or behaviors but caused a significant reduction of gait, clasping, and tremor behaviors in Hdh150Q/150Q mice. Rhes deletion did not affect the loss of striatal DARPP-32 protein levels but abrogated the hyper ribosomal protein S6 kinase beta-1 (S6K) phosphorylation, which is a substrate for a mechanistic target of rapamycin complex 1 (mTORC1) signaling, in Hdh(CAG)150 mice. Interestingly, striatal Rhes protein levels were downregulated in the striatum of Hdh(CAG)150 mice, indicating a potential compensatory mechanisms at work. Thus, Rhes deletion prevents age-dependent behavioral deficits and diminishes hyperactive mTORC1-S6K signaling in Hdh(CAG)150 knock-in mice HD striatum.

neuroscience↗

bioRxiv2020

Bacterial single-cell genomics enables phylogenetic analysis of in vitro evolutionary studies

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer, but have not yet been deployed to study evolving microbial populations. Here, we present an approach for single bacterial genomic analysis using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to in vitro experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from the population we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to both develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.

genomics↗

bioRxiv2020

A Role for Maternal Factors in Suppressing Cytoplasmic Incompatibility

Wolbachia are maternally transmitted bacterial endosymbionts, carried by approximately half of all insect species. Wolbachia prevalence in nature stems from manipulation of host reproduction to favor the success of infected females. The best known reproductive modification induced by Wolbachia is referred to as sperm-egg Cytoplasmic Incompatibility (CI). In CI, the sperm of Wolbachia-infected males cause embryonic lethality, attributed to paternal chromatin segregation defects during early mitotic divisions. Remarkably, the embryos of Wolbachia-infected females "rescue" CI lethality, yielding egg hatch rates equivalent to uninfected female crosses. Several models have been discussed as the basis for Rescue, but none have been demonstrated in robust CI models, which are genetically intractable. As such, the extent of host involvement in Rescue remains untested. In this study, we used a chemical feeding approach to assess maternal contributions to CI suppression in Drosophila simulans. We found that uninfected females exhibited significantly higher CI egg hatch rates in response to seven chemical treatments that affect DNA integrity, cell cycle control, and protein turnover. Three of these treatments suppressed CI induced by endogenous wRi Wolbachia, as well as an ectopic wMel Wolbachia infection. When framed in terms of existing literature, the results implicate DNA integrity as a dynamic aspect of CI suppression for different Wolbachia strains. The framework presented here, applied to diverse CI models, will further enrich our understanding of host reproductive manipulation by insect endosymbionts.

microbiology↗

bioRxiv2020

Robust Production of Uniform Human Cerebral Organoids from Pluripotent Stem Cells

Human cerebral organoid (hCO) models offer the opportunity to understand fundamental processes underlying human specific cortical development and pathophysiology in an experimentally tractable system. While diverse methods to generate brain organoids have been developed, a major challenge has been the production of organoids with reproducible cell type heterogeneity and macroscopic morphology. Here, we directly addressed this problem by establishing a robust production pipeline to generate morphologically consistent (ie uniform) hCOs and achieve a success rate of >80%. These hCOs include both a radial glial stem cell compartment and electrophysiologically competent mature neurons. Moreover, we show using immunofluorescence microscopy and single cell profiling, that individual organoids display reproducible cell type compositions that are conserved upon extended culture. We expect that application of this method will provide new insights into brain development and disease processes.

neuroscience↗

bioRxiv2020

Microbial competition reduces interaction distances to the low {micro}m-range

Metabolic interactions between cells affect microbial community compositions and hence their function in ecosystems. It is well-known that under competition for the exchanged metabolite, concentration gradients constrain the distances over which interactions can occur. However, interaction distances are typically quantified in two-dimensional systems or without accounting for competition or other metabolite-removal, conditions which may not very often match natural ecosystems. We here analyze the impact of cell-to-cell distance on unidirectional cross-feeding in a three-dimensional system with competition for the exchanged metabolite. Effective interaction distances were computed with a reaction-diffusion model and experimentally verified by growing a synthetic consortium of 1 {micro}m-sized metabolite producer, receiver and competitor cells in different spatial structures. We show that receivers cannot interact with producers [~]15 {micro}m away from them, as product concentration gradients flatten close to producer cells. We developed an aggregation protocol and created variants of the receiver cells import system, to show that within producer-receiver aggregates even low affinity receiver cells could interact with producers. These results show that competition or other metabolite-removal of a public good in a three-dimensional system reduces the interaction distance to the low micrometer-range, highlighting the importance of concentration gradients as physical constraint for cellular interactions.

microbiology↗

bioRxiv2019

Efficient whole genome sequencing of influenza A viruses

The constant threat of emergence for novel pathogenic influenza A viruses with pandemic potential, makes full-genome characterization of circulating influenza viral strains a high priority, allowing detection of novel and re-assorting variants. Sequencing the full-length genome of influenza A virus traditionally required multiple amplification rounds, followed by the subsequent sequencing of individual PCR products. The introduction of high-throughput sequencing technologies has made whole genome sequencing easier and faster. We present a simple protocol to obtain whole genome sequences of hypothetically any influenza A virus, even with low quantities of starting genetic material. The complete genomes of influenza A viruses of different subtypes and from distinct sources (clinical samples of pdmH1N1, tissue culture-adapted H3N2 viruses, or avian influenza viruses from cloacal swabs) were amplified with a single multisegment reverse transcription-PCR reaction and sequenced using Illumina sequencing platform. Samples with low quantity of genetic material after initial PCR amplification were re-amplified by an additional PCR using random primers. Whole genome sequencing was successful for 66% of the samples, whilst the most relevant genome segments for epidemiological surveillance (corresponding to the hemagglutinin and neuraminidase) were sequenced with at least 93% coverage (and a minimum 10x) for 98% of the samples. Low coverage for some samples is likely due to an initial low viral RNA concentration in the original sample. The proposed methodology is especially suitable for sequencing a large number of samples, when genetic data is urgently required for strains characterization, and may also be useful for variant analysis.

microbiology↗

bioRxiv2018

Integrin beta1 optimizes diabetogenic T cell migration and function in the pancreas

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas during autoimmune diabetes remains unclear. Using intra-vital 2-photon microscopy in a mouse model of diabetes, we found that CXCR3 chemokine downregulated CD8+ T cell motility specifically within islets, promoting effector cell confinement to their target sites. In contrast, T cell velocity and directionality in the exocrine tissue were enhanced along blood vessels and extra-cellular matrix fibers. This guided migration implicated integrin-dependent interactions, since integrin blockade impaired exocrine T cell motility. In addition, integrin {beta}1 blockade decreased CD4+ T cell effector phenotype specifically in the pancreas. Thus, we unveil an important role for integrins in the pancreas during autoimmune diabetes that may have important implications for the design of new therapies.

immunology↗