Biology subjects

Day, L. Z.

Publications and source records attributed to Day, L. Z..

5 recordsLinked to original sources

bioRxiv2026

Astrocyte Reactivity by Alcohol Dependence in the Central Amygdala

Astrocytes play essential roles in maintaining brain homeostasis and in contributing to synaptic functions, but, in response to injury, infection, or disease, astrocytes can downregulate their homeostatic and physiological functions while increasing neuroinflammatory responses. The central amygdala (CeA) is important for stress responsivity and the development of alcohol (ethanol) dependence. Using a multi-omics approach in Aldh1l1-EGFP/Rpl10a mice and the chronic intermittent ethanol two-bottle choice (CIE-2BC) model, we have characterized the translational response of CeA astrocytes, as well as the proteomic and phosphoproteomic changes in ethanol dependent, non-dependent, and naive mice. We identified astrocyte-specific alterations in neuroimmune functions and antioxidant/oxidative stress pathways in ethanol dependent mice as well as cytoskeletal plasticity related pathways in non-dependent mice. Proteomic analysis showed down-regulation of astrocyte physiological functions in dependent animals while phosphoproteomic analysis identified pathways associated with cytoskeleton remodeling in both dependent and non-dependent mice. Reconstructions of astrocyte morphologies demonstrated increased CeA astrocyte complexity in dependent and non-dependent groups compared to naive mice. The astrocyte-specific activation of neuroimmune and antioxidant pathways, down-regulation of homeostatic functions, alteration in protein phosphorylation-mediated cytoskeleton remodeling, and increased astrocyte morphological complexity demonstrate that ethanol dependence induces astrocyte reactivity in the CeA consistent with both adaptive and maladaptive changes. These findings highlight the role of CeA astrocytes in the progression from alcohol intake to dependence and represent a first step toward identifying astrocyte-specific therapeutic strategies to treat Alcohol Use Disorder (AUD) aimed at potentiating reactive astrocyte adaptive changes and inhibiting maladaptive responses.

neuroscience↗

bioRxiv2022

Proteome mapping of the human pancreatic islet microenvironment reveals endocrine-exocrine signaling sphere of influence

The need for a clinically accessible method with the ability to match protein activity within heterogeneous tissues is currently unmet by existing technologies. Our proteomics sample preparation platform, named microPOTS (Microdroplet Processing in One pot for Trace Samples), can be used to measure relative protein abundance in micron-scale samples alongside the spatial location of each measurement, thereby tying biologically interesting proteins and pathways to distinct regions. However, given the smaller sample number and amount of tissue measu red, standard mass spectrometric analysis pipelines have proven inadequate. Here we describe how existing computational approaches can be adapted to focus on the specific biological questions asked in spatial proteomics experiments. We apply this approach to present an unbiased characterization of the human islet microenvironment comprising the entire complex array of tissues involved while maintaining spatial information and the degree of the islets sphere of influence. We identify specific functional activity unique to the pancreatic islet cells and demonstrate how far their signature can be measured. Our results show that we can distinguish pancreatic islet cells from the neighboring exocrine tissue environment, recapitulate known biological functions of islet cells, and identify a spatial gradient in the expression of RNA processing proteins within the islet microenvironment.

systems biology↗

bioRxiv2021

Mutagenesis of human cytomegalovirus glycoprotein L disproportionately disrupts gH/gL/gO over gH/gL/pUL128-131.

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but the how the gH/gL portion of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB dependent cell-cell fusion, but were still able to form gH/gL/pUL128-131 and induce receptor-interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. Effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFR, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a "hyperactive" gH/gL/gO. Recently published crystallography and cryo-EM studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCEThe endemic beta-herpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes and may provide a basis for more refined evaluations of neutralizing antibodies.

microbiology↗

bioRxiv2019

Naturally Occurring Polymorphisms in Human Cytomegalovirus gO Exert Epistatic Influences on Cell-Free and Cell-To-Cell Spread, and Antibody Neutralization on gH Epitopes.

The human cytomegalovirus (HCMV) glycoproteins H and L (gH/gL) can be bound by either gO, or the UL128-131 proteins to form complexes that facilitate entry and spread and the complexes formed are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131 and this can impact infectivity and cell tropism. In this report, we investigated how natural interstrain variation in the amino acid sequence of gO influences the biology of HCMV. Heterologous gO recombinants were constructed in which 6 of the 8 alleles or genotypes (GT) of gO were analyzed in the backgrounds of strain TR and Merlin (ME). The levels of gH/gL complexes were not affected, but there were impacts on entry, spread and neutralization by anti-gH antibodies. AD169 (AD) gO (GT1a) drastically reduced cell-free infectivity of both strains on fibroblasts and epithelial cells. PHgO(GT2a) increased cell-free infectivity of TR in both cell types, but spread in fibroblasts was impaired. In contrast, spread of ME in both cell types was enhanced by Towne (TN) gO (GT4), despite similar cell-free infectivity. TR expressing TNgO(GT4) was resistant to neutralization by anti-gH antibodies AP86 and 14-4b, whereas ADgO(GT1a) conferred resistance to 14-4b, but enhanced neutralization by AP86. Conversely, ME expressing ADgO(GT1a) was more resistant to 14-4b. These results suggest; 1) mechanistically distinct roles for gH/gL/gO in cell-free and cell-to-cell spread, 2) gO isoforms can differentially shield the virus from neutralizing antibodies, and 3) effects of gO polymorphisms are epistatically dependent on other variable loci. IMPORTANCEAdvances in HCMV population genetics have greatly outpaced understanding of the links between genetic diversity and phenotypic variation. Moreover, recombination between genotypes may shuffle variable loci into various combinations with unknown outcomes. UL74(gO) is an important determinant of HCMV infectivity, and one of the most diverse loci in the viral genome. By analyzing interstrain heterologous UL74(gO) recombinants, we show that gO diversity can have dramatic impacts on cell-free and cell-to-cell spread as well as on antibody neutralization and that the manifestation of these impacts can be subject to epistatic influences of the global genetic background. These results highlight the potential limitations of laboratory studies of HCMV biology that use single, isolated genotypes or strains.

microbiology↗

bioRxiv2019

Specialization to cell-free or cell-associated spread by BAC-cloned HCMV strains not determined by the UL128-131 and RL13 loci

It is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell-associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The BAC-clone Merlin (ME), expresses abundant UL128-131, is RL13-impaired and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, RL13-intact and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread, but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptional repressed, cell-to-cell spread of ME was still more efficient than TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci. IMPORTANCEBoth cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether "cell-associated" phenotypes are simply the result of poor cell-free spread, or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

microbiology↗