Biology subjects

Pham, K.

Publications and source records attributed to Pham, K..

35 records · Page 2Linked to original sources

bioRxiv2024

Proteome-wide reverse molecular docking reveals folic acid receptor as a mediator of PFAS-induced neurodevelopmental toxicity

Per- and polyfluoroalkyl substances (PFAS) are a class of long-lasting chemicals with widespread use and environmental persistence that have been increasingly studied for their detrimental impacts to human and animal health. Several major PFAS species are linked to neurodevelopmental toxicity. For example, epidemiological studies have associated prenatal exposure to perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) with autism risk. However, the neurodevelopmental toxicities of major PFAS species have not been systematically evaluated in an animal model, and the molecular mechanisms underlying these toxicities have remained elusive. Using a high-throughput zebrafish social behavioral model, we screened six major PFAS species currently under regulation by the Environmental Protection Agency (EPA), including PFOA, PFNA, perfluorooctane sulfonate (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorobutane sulfonate (PFBS), and hexafluoropropylene oxide dimer acid ammonium salt (GenX). We found that embryonic exposure to PFNA, PFOA, and PFOS induced social deficits in zebrafish, recapitulating one of the hallmark behavioral deficits in autistic individuals. To uncover protein targets of the six EPA-regulated PFAS, we screened a virtual library containing predicted binding pockets of over 80% of the 3D human proteome through reverse molecular docking. The screen predicts that folate receptor beta (FR-{beta}, encoded by the gene FOLR2) interacts strongly with PFNA, PFOA, and PFOS but to a lesser degree with PFHxS, PFBS, and GenX, correlating positively with their in vivo toxicity. These predictions were validated through in silico molecular docking, in vitro protein binding analysis, and in vivo loss-of-function verification. Furthermore, embryonic co-exposure to folic acid effectively rescued social deficits induced by PFAS. The folate pathway has been implicated in autism, indicating a novel molecular mechanism for PFAS in autism etiology.

pharmacology and toxicology↗

bioRxiv2024

Late-life short-term dietary restriction ameliorates intestinal stem cell function and impacts intestinal stem cell DNA methylation

Aging reduces the function of intestinal stem cells (ISCs) and dietary restriction (DR) has been shown to improve ISC function in young mice. However, there are no studies to-date that has evaluated the effect of short-term DR (stDR) implemented late-in-life on ISC function. Data from our laboratory shows for the first time that stDR can ameliorate the age-related decline in ISC function by in-vitro enteroid forming assay. Transcriptomic analysis of the Lgr5+ ISCs identified genes not commonly known for ISC function but showed a robust change with age which were reversed by stDR. Evaluation of the methylation status of selected genes (Slc28a3, Ly9, Cd2, Platr4) showed association between changes in 5mCG levels in the cis-acting regions of the genes and their expression. Our data suggest a potential role for DNA methylation in stDRs effect on ISC function.

molecular biology↗

bioRxiv2024

Transcriptomic and multi-scale network analyses reveal key drivers of cardiovascular disease

Cardiovascular diseases (CVDs) and pathologies are often driven by changes in molecular signaling and communication, as well as in cellular and tissue components, particularly those involving the extracellular matrix (ECM), cytoskeleton, and immune response. The fine-wire vascular injury model is commonly used to study neointimal hyperplasia and vessel stiffening, but it is not typically considered a model for CVDs. In this paper, we hypothesize that vascular injury induces changes in gene expression, molecular communication, and biological processes similar to those observed in CVDs at both the transcriptome and protein levels. To investigate this, we analyzed gene expression in microarray datasets from injured and uninjured femoral arteries in mice two weeks post-injury, identifying 1,467 significantly and differentially expressed genes involved in several CVDs such as including vaso-occlusion, arrhythmia, and atherosclerosis. We further constructed a protein-protein interaction network with seven functionally distinct clusters, with notable enrichment in ECM, metabolic processes, actin-based process, and immune response. Significant molecular communications were observed between the clusters, most prominently among those involved in ECM and cytoskeleton organizations, inflammation, and cell cycle. Machine Learning Disease pathway analysis revealed that vascular injury-induced crosstalk between ECM remodeling and immune response clusters contributed to aortic aneurysm, neovascularization of choroid, and kidney failure. Additionally, we found that interactions between ECM and actin cytoskeletal reorganization clusters were linked to cardiac damage, carotid artery occlusion, and cardiac lesions. Overall, through multi-scale bioinformatic analyses, we demonstrated the robustness of the vascular injury model in eliciting transcriptomic and molecular network changes associated with CVDs, highlighting its potential for use in cardiovascular research.

genomics↗

bioRxiv2024

Large-scale genomic analysis of SARS-CoV-2 Omicron BA.5 emergence in the United States

The COVID-19 pandemic is marked by the continuing emergence of novel SARS-CoV-2 variants. Questions remain about the mechanisms with which these lineages establish themselves in new geographical areas. In this study, we performed a discrete phylogeographic analysis on [~]19,000 SARS-CoV-2 sequences of Omicron BA.5 lineages between February and June 2022 to better understand how it emerged in different regions of the United States (U.S.). We found that the earliest introductions came from Africa, the putative origin of the variant, but the majority were from Europe, correlating with the high volume of air travelers. Additionally, the analysis revealed extensive domestic transmission between different regions of the U.S. driven by population size and cross-country transmission. Results suggest that most of the within-U.S. spread was between three regions that include California, New York, and Florida. Our results form a framework for understanding novel SARS-CoV-2 variant emergence in the U.S.

genomics↗

bioRxiv2024

FISHnet: Detecting chromatin domains in single-cell sequential Oligopaints imaging data

Sequential Oligopaints DNA FISH is an imaging technique that measures higher-order genome folding at single-allele resolution via multiplexed, probe-based tracing. Currently there is a paucity of algorithms to identify 3D genome features in sequential Oligopaints data. Here, we present FISHnet, a graph theory method based on optimization of network modularity to detect chromatin domains and boundaries in pairwise distance matrices. FISHnet uncovers cell type-specific domain-like folding patterns on single alleles, thus enabling future studies aiming to elucidate the role for single-cell folding variation on genome function.

molecular biology↗

bioRxiv2024

Multiplexed optical barcoding and sequencing for spatial omics

Spatial omics has brought a fundamental change in the way that we study cell and tissue biology in health and disease. Among various spatial omics methods, genome-scale imaging allows transcriptomic, 3D-genomic, and epigenomic profiling of individual cells with high spatial (subcellular) resolution but often requires a preselection of targeted genes or genomic loci. On the other hand, spatially dependent barcoding of molecules followed by sequencing provides untargeted, genome-wide profiling but typically lacks single-cell resolution. Here, we report a spatial omics method that could potentially combine the power of the two approaches by optically controlled spatial barcoding followed by sequencing. Specifically, we utilize patterned light to encode the locations of cells in tissues using oligonucleotide-based barcodes and then identify the barcoded molecular content, such as mRNAs, by sequencing. This optical barcoding method is designed with multiplexing and error-correction capacity and achieved by a light-directed ligation chemistry that attaches distinct nucleic-acid sequences to the reverse transcribed cDNA products at different locations. As a proof of principle for this method, we demonstrated high-efficiency in situ light-directed ligation, spatially dependent barcoding with multiplexed light-controlled ligations, and high-accuracy detection of spatially barcoded mRNAs in cells.

genomics↗

bioRxiv2023

Consistent specificity and efficiency of tamoxifen-mediated cre induction across ages

Temporally controlling cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report Tam induction at early post-natal/juvenile (<2 m.o.) mouse ages, but age-related neurodegeneration and aging studies can require cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam mediated cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at 3-6 m.o. or 20 m.o. of age. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each cre and flox mouse line should be validated independently, however, these findings demonstrate that Tam-mediated cre induction can be performed even into older mouse ages.

genomics↗

bioRxiv2023

Microglial MHC-I induction with aging and Alzheimer's is conserved in mouse models and humans

Major Histocompatibility Complex I (MHC-I) CNS cellular localization and function is still being determined after previously being thought to be absent from the brain. MHC-I expression has been reported to increase with brain aging in mouse, rat, and human whole tissue analyses but the cellular localization was undetermined. Neuronal MHC-I is proposed to regulate developmental synapse elimination and tau pathology in Alzheimers disease (AD). Here we report that across newly generated and publicly available ribosomal profiling, cell sorting, and single-cell data, microglia are the primary source of classical and non-classical MHC-I in mice and humans. Translating Ribosome Affinity Purification-qPCR analysis of 3-6 and 18-22 month old (m.o.) mice revealed significant age-related microglial induction of MHC-I pathway genes B2m, H2-D1, H2-K1, H2-M3, H2-Q6, and Tap1 but not in astrocytes and neurons. Across a timecourse (12-23 m.o.), microglial MHC-I gradually increased until 21 m.o. and then accelerated. MHC-I protein was enriched in microglia and increased with aging. Microglial expression, and absence in astrocytes and neurons, of MHC-I binding Leukocyte Immunoglobulin-like (Lilrs) and Paired immunoglobin-like type 2 (Pilrs) receptor families could enable cell-autonomous MHC-I signaling and increased with aging in mice and humans. Increased microglial MHC-I, Lilrs, and Pilrs were observed in multiple AD mouse models and human AD data across methods and studies. MHC-I expression correlated with p16INK4A, suggesting an association with cellular senescence. Conserved induction of MHC-I, Lilrs, and Pilrs with aging and AD opens the possibility of cell-autonomous MHC-I signaling to regulate microglial reactivation with aging and neurodegeneration.

neuroscience↗

bioRxiv2022

Hypothalamic melanocortin-4 receptors on astrocytes mediate inflammation and body weight homeostasis

World-wide, nearly 40% of the adult population is classified as clinically obese. Central inflammation is highly correlated with obesity and increases morbidity and deterioration of health. The hypothalamus is a brain region that governs many facets of energy homeostasis, and melanocortins in the hypothalamus both decrease feeding and increase metabolism via melanocortin-4 receptors (MC4Rs). Although MC4Rs are present on neurons and astrocytes (aMC4R) previous work has focused almost exclusively on the neuronal population with the contribution of aMC4R on these processes largely unknown. Our objective was to determine the effects of hypothalamic aMC4R deletion on central and peripheral inflammation, as well as feeding and body weight homeostasis. Adult MC4R fl/fl mice were microinjected with an astrocyte-specific promoter driving Cre-expression (AAV-GFAP-GFP-Cre) or AAV-control (AAV-GFAP-GFP; n=4-7/group/sex) to produce a hypothalamic knock-down of aMC4R (KD). Body weight and composition were monitored throughout the study, and indirect calorimetry was conducted at 1 and 4 weeks after AAV injection. Acquisition of operant self-administration of palatable food was also examined. Mice were euthanized 7-8 weeks post AAV injection and brain and tissue samples were collected. We observed a significant increase in body weight, feeding, and energy balance in the KD group compared to control group. Inflammation was significantly increased centrally in KD mice within the hypothalamus, but not peripherally within serum. Additionally, aMC4R KD mice trended towards an increased reward learning for palatable food. This is the first demonstration that hypothalamic aMC4R, independent of neuronal MC4R, is important in modulating inflammation as well as contributing to energy balance. These results provide an integral understanding of the aMC4R system that will provide the foundation for future studies investigating the role of aMC4R in various disease states.

neuroscience↗

bioRxiv2022

An unexpected histone chaperone function for the MIER1 histone deacetylase complex

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and to deposit histone octamer onto nucleosome-depleted regions of DNA.

biochemistry↗

bioRxiv2021

Single-cell analysis reveals MHCII expressing keratinocytes in pressure ulcers with worse healing outcomes

Pressure ulcer (PU) is a chronic wound often seen in spinal cord injury patients and other bed-bound individuals, particularly in the elderly population. Despite its association with high mortality, the pathophysiology of PU remains poorly understood. Here, we compared single-cell transcriptomic profiles of human epidermal cells from PU wound edges with those from uninjured skin and acute wounds (AWs) in healthy donors. We identified significant shifts in the cell composition and gene expression patterns in PU. In particular, we found that major histocompatibility complex class II (MHCII) expressing keratinocytes were enriched in patients with worse healing outcomes. Furthermore, we showed that the IFN{gamma} in PU-derived wound fluid could induce MHCII expression in keratinocytes and that these wound fluid-treated keratinocytes inhibited autologous T cell activation. In line with this observation, we found that T cells from PUs enriched with MHCII+ keratinocytes produced fewer inflammatory cytokines. Overall, our study provides a high-resolution molecular map of human PU compared to AW and intact skin, providing new insights into PU pathology and the future development of tailored wound therapy.

genomics↗

bioRxiv2020

Electronic cigarettes induce mitochondrial DNA damage and trigger toll-like receptor 9-mediated atherosclerosis

ObjectiveBoth electronic cigarette (e-cig) use and toll-like receptor 9 (TLR9) activation have been implicated in promoting atherosclerosis. In this study we aimed to investigate the causative relationship of e-cig exposure on TLR9 activation and atherosclerosis development. Approach and ResultsEight-week-old ApoE-/- mice fed normal chow diet were exposed to e-cig vapor (ECV) for 2 h/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining of aortic root and greatly upregulated TLR9 expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by upregulated TLR9 expression in human femoral artery atherosclerotic plaques in e-cig smokers. Intriguingly, we found a significant increase of damaged mitochondria DNA level in the circulating blood of ECV exposed mice. Furthermore, administration of TLR9 antagonist prior to ECV exposure not only alleviated atherosclerotic lesion and the upregulation of TLR9 in plaques, but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ classical monocytes. Surprisingly, we found that the cytoplasmic mtDNA isolated from ECV extract-treated cells can greatly enhance the expression of TLR9 in reporter cells. ConclusionE-cig induces mtDNA damage and the mtDNA in circulating blood stimulates the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacologic target of ECV-related inflammation and cardiovascular diseases.

pharmacology and toxicology↗

bioRxiv2019

Developing T cells form an immunological synapse for passage through the β-selection checkpoint

The {beta}-selection checkpoint of T cell development tests whether the cell has recombined its genomic DNA to produce a functional T Cell Receptor {beta} (TCR{beta}) receptor. Passage through the {beta}-selection checkpoint requires the nascent TCR{beta} protein to mediate signaling through a pre-TCR complex. In this study, we show that developing T cells at the {beta}-selection checkpoint establish an immunological synapse in in vitro & in situ, resembling that of the mature T cell. The immunological synapse is dependent on two key signaling pathways known to be critical for the transition beyond the {beta}-selection checkpoint, Notch and CXCR4 signaling. In vitro and in situ analyses indicate that the immunological synapse promotes passage through the {beta}-selection checkpoint. Collectively, these data indicate that developing T cells regulate pre-TCR signaling through the formation of an immunological synapse. This signaling platform integrates cues from Notch, CXCR4, and MHC on the thymic stromal cell, to allow transition beyond the {beta}-selection checkpoint.\n\nSummaryT cell development requires testing whether genomic rearrangement has produced a T cell receptor capable of transmitting signals. Most T cells fail this test. Here, we show that passage through the {beta}-selection checkpoint requires assembly of a platform to support TCR signaling.

immunology↗

bioRxiv2019

A new role for Notch in control of polarity and asymmetric cell division of developing T cells

A fundamental question in biology is how single cells can reliably produce progeny of different cell types. Notch signalling frequently facilitates fate determination. Asymmetric cell division (ACD) often controls segregation of Notch signalling by imposing unequal inheritance of regulators of Notch. Here, we assessed the functional relationship between Notch and ACD in mouse T cell development. To attain immunological specificity, developing T cells must pass through a pivotal stage termed {beta}-selection, which involves Notch signalling and ACD. We assessed functional interactions between Notch and ACD during {beta}-selection using direct presentation of Notch ligands, DL1 and DL4, and pharmacological inhibition of Notch signalling. Contrary to prevailing models, we find Notch controls distribution of Notch1 itself and cell fate determinants, -Adaptin and Numb. Notch and CXCR4 signalling cooperated to drive polarity during division. Thus, Notch signalling directly orchestrates ACD, and Notch1 is differentially inherited by sibling cells.

cell biology↗

bioRxiv2019

PIK3CA in KrasG12D/Trp53R172H Tumor Cells Promotes Immune Evasion by Limiting Infiltration of T Cells in a Model of Pancreatic Cancer

The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapies. Here we show using a syngeneic orthotopic implantation model of pancreatic cancer that Pik3ca regulates tumor immunogenicity. Genetic silencing of Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors leads to infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progress and kill all of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminates Pik3ca-null tumors in immunodeficient mice. Loss of PIK3CA or inhibition of its effector, AKT, increases the expression of MHC Class I and CD80 on tumor cells. These changes contribute to the increased susceptibility of Pik3ca-null tumors to T cell surveillance. These results indicate that tumor cell PIK3CA-AKT signaling limits T cell recognition and clearance of pancreatic cancer cells. Strategies that target this pathway may yield an effective immunotherapy for this cancer.\n\nSIGNIFICANCEPIK3CA-AKT signaling in pancreatic cancer cells limits T cell infiltration and clearance of tumors by suppressing the surface expression of MHC Class I and CD80. Targeting the PIK3CA-AKT pathway in tumor cells provides a new avenue for discovery of novel pancreatic cancer immunotherapies.

cancer biology↗

bioRxiv2017

Dynamics of the upper airway microbiome in the pathogenesis of asthma-associated persistent wheeze in preschool children

Repeated cycles of infection-associated lower airway inflammation drives the pathogenesis of persistent wheezing disease in children. Tracking these events across a birth cohort during their first five years, we demonstrate that >80% of infectious events indeed involve viral pathogens, but are accompanied by a shift in the nasopharyngeal microbiome (NPM) towards dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change in NPM frequently precedes the appearance of viral pathogens and acute symptoms. In non-sensitized children these events are associated only with \"transient wheeze\" that resolves after age three. In contrast, in children developing early allergic sensitization, they are associated with ensuing development of persistent wheeze, which is the hallmark of the asthma phenotype. This suggests underlying pathogenic interactions between allergic sensitization and antibacterial mechanisms.

genetics↗

bioRxiv2016

Genomic analysis of Mycobacterium tuberculosis reveals complex etiology of tuberculosis in Vietnam including frequent introduction and transmission of Beijing lineage and positive selection for EsxW Beijing variant

Introduction Introduction Accession codes AUTHOR CONTRIBUTIONS COMPETING FINANCIAL INTERESTS Online Methods References Tuberculosis (TB) is a leading cause of death from infectious disease and the global burden is now higher than at any point in history 1,2 Despite coordinated efforts to control TB transmission, the factors contributing to its successful spread remain poorly understood. Vietnam is identified as one of 30 high burden countries for TB and MDR-TB with an incidence of 137 TB cases per 100,000 individuals in 2015 2 Recent phylogenomic analyses of the causative agent Mycobacterium tuberculosis (Mtb) in other high-prevalence regions have provided insights into the complex processes underlying TB transmission ...

genomics↗