Biology subjects

King, D.

Publications and source records attributed to King, D..

At least 19 recordsLinked to original sources

bioRxiv2026

APOA1 links a late fetal epithelial program to colonic maturation and injury responses

Developmental programs establish tissue identity and organization, but how their reactivation contributes to adult tissue responses remains poorly understood. Here, we identify a functional contribution of apolipoprotein A-I (APOA1), which marks a late fetal colonic epithelial state that reappears after injury. Spatial and temporal profiling localized Apoa1 transcription predominantly to epithelial cells at the tips of developing proximal colonic folds. Apoa1 loss delayed fold maturation and altered the abundance and composition of developing enteroendocrine populations, linking this apolipoprotein to mucosal architecture and epithelial differentiation. APOA1 protein closely interacted with WNT5A during development and injury, while Apoa1 deficiency increased Wnt5a expression and altered its epithelial protein distribution. Following colonic injury, single-cell and spatial profiling identified genotype-associated transcriptional changes involving stress responses, barrier and immune functions, and metabolism, with a shared component across males and females. These changes accompanied reduced enteroendocrine representation and increased representation of inflammation-associated macrophages. Finally, regional and compartmental expression analyses, together with hindgut explant perturbation, identified HOXB7 transcription factor as a regulator of developmental Apoa1 expression. Together, these findings establish functional relevance for a component of a reactivated late fetal epithelial program, connecting APOA1 to colonic maturation and the epithelial and inflammatory organization of injured tissue.

molecular biology↗

bioRxiv2026

A 50-marker mass cytometry panel to expand analysis of the functional breadth of human immune cells

Human immune single-cell proteomic functional profiling has historically been performed with a limited number of inflammatory and/or cytotoxic readouts, capturing only a fraction of the complex orchestra of factors that comprise immune responses. Given the rising global crisis of chronic inflammation and the lack of clinically available treatment options, there is an urgent need to gain insight into the cell subsets that exhibit anti-inflammatory functional profiles and elucidate the mechanisms regulating these effector capacities. To address this, we developed a 50-marker CyTOF panel that enables unprecedented functional fingerprinting of human T cells, NK cells, monocytes, and B cells, detecting 24 intracellular targets. Healthy donor PBMCs were stimulated ex vivo and stained with this panel; from T cells, cytokines associated with the hallmark Type 1 (IFN-{gamma}, TNF-), Type 2 (IL-4, IL-5, and IL-13), and Type 17 (IL-17A, IL-17F) functional lineages were detected, as well as the chemokines MIP-1-, MIP1-{beta}, and IL-8 and the cell repair factor amphiregulin; from monocytes, IL-1{beta}, IL-35, and IL-8 were detected. To ascertain if some of the cytokines less commonly included in Intracellular Cytokine Staining (ICS) panels were produced in response to physiological TCR stimulation via viral peptides, we measured the T cell response to a CMV-EBV-Flu (CEF) pool; in addition to TNF-, IFN-{gamma}, and IL-2, we also found that individual T cells produced additional cytokines with IFN-{gamma} and TNF-, such as amphiregulin, MIP-1, IL-13, and IL-4. This mass cytometry panel provides an exceptionally broad and deeply resolved view of the functional diversity of human immune cells, surpassing, to our knowledge, the capabilities of previously reported approaches. Due to minimal signal overlap, CyTOF enables flexible panel customization, allowing markers and metal tags to be readily expanded or modified. Based on its resolution and adaptability, we anticipate that this panel and its derivatives will enable the discovery of novel immunomodulatory mechanisms for therapeutic intervention.

immunology↗

bioRxiv2025

Superior intracellular detection of cytokines, transcription factors, and phosphoproteins by CyTOF compared with fluorescent cytometry

Unraveling biological complexity, whether it be immune subset distribution in infectious disease(s), autoimmunity or tumor heterogeneity, requires technologies capable of single-cell proteomic analysis, such as flow cytometry. Surface immunophenotyping alone is often insufficient, as interrogating functional capacity is required to determine cellular mechanisms and effectively inform diagnostic biomarker discovery, therapeutics and vaccine development. However, large panels with intracellular markers are subject to numerous challenges, including spectral overlap and background cellular autofluorescence, reducing resolving power for rare subsets or populations defined by low-abundance expression. We posited that mass cytometry may overcome such limitations; to address this, three small (11-12-plex) clone-matched antibody panels were evaluated by spectral flow and mass cytometry. Panels were comprised of surface and intracellular targets (phospho-epitopes, transcription factors or cytokines) and designed to minimize fluorescence spectral overlap. CyTOF technology offered superior signal resolution across the range of intracellular targets. Improved signal-to-noise provided better resolution of phospho-events and transcription factor expression, in particular TOX and T-bet. Most strikingly, stimulation-specific IL-10+ and IL-13+ cells were only detected by CyTOF. Superior resolution of these cytokines enabled accurate population clustering, permitting more unique immune cell signatures to be found, including Tr1 and Tc2 populations, thus providing a more comprehensive picture of the immuno-diversity present. Our findings indicate that CyTOF technology could catalyze seminal discoveries in functional immune profiling, driving therapeutic design and diagnostics.

immunology↗

bioRxiv2025

The SRG RAT supports human cell xenotransplantation through enhanced tumor microenvironment interactions

The use of immunodeficient mice for human tumor engraftment is an essential model of human cancer, with uses ranging from basic science to translational research. However, low engraftment rates, slow growth, and smaller tumor volumes can be limitations. Previously, we reported a highly immunodeficient rat strain with the functional deletion of both the Rag2 and Il2rg genes on the Sprague-Dawley background (SRG RAT(R)), which lacks B, T, and NK cells. Here, we subcutaneously engrafted two cell-derived xenograft (CDX) and seven patient-derived xenograft (PDX) models, including prostate, lung, ovarian, and uterine cancer models, into the SRG rat or NSG mouse models and tracked tumor growth. In all cases, the engraftment and tumor growth rates were better supported in the SRG rat compared to the NSG mouse. Interestingly, the SRG rat is not more immunocompromised than the NSG mouse, suggesting alternative mechanisms leading to the supportive growth in the SRG rat. Therefore, we explored potential differences in the tumor microenvironment (TME) between models grown in the two host animals. Lung PDX models grown in SRG rats showed enhanced formation of vasculature and stroma and were morphologically more consistent with the originating patient tumors. IHC analysis of the NCI-H660 CDX model showed differences in the tumors stroma, vasculature, and macrophages when grown in the two host species. Single-cell spatial imaging of engrafted tumors showed upregulation of the human CXCL2 and TCIM in NCI-H660 tumors grown in the SRG rat versus the NSG mouse, both of which have been linked to poor prognosis in cancer. Combined, our data demonstrate that the SRG rat supports the growth of multiple human cancer types and displays enhanced tumor microenvironment interactions compared to NSG mice.

cancer biology↗

bioRxiv2025

Synergistic MAPT mutations as a platform to uncover modifiers of tau pathogenesis

The natively unfolded tau (MAPT) protein is extremely soluble, which poses challenges when modeling neurofibrillary tangle (NFT) pathology in Alzheimers disease (AD). To overcome this hurdle, we combined P301L and S320F mutations (PL-SF) to generate a rapid and reliable tau pathology platform to expedite the discovery of factors that modify tau aggregation. Using this model, we evaluated heat-shock proteins (Hsp), which have been linked to tau pathology, but whose role in AD remains enigmatic and controversial. In primary neurons, expression of Hsp70, but not Hsc70 or Hsp90, exacerbated tau aggregation. Conversely, lowering Hsp70 or employing a chaperone-deficient tau mutant (PL-SF-4{Delta}) reduced tau phosphorylation and abrogated tau aggregation, highlighting Hsp70 as a key driver of tau aggregation. Hsp70 foci clustered within and surrounding neuritic plaques and NFTs in post-mortem AD brain. Functionally, mature aggregate-bearing neurons showed deficits in neuronal firing and network communication, which were restored by chaperone-binding deficient tau variants that abrogated tau pathology. This study provides a powerful cell-intrinsic model for accelerated tau aggregation, which can be harnessed to identify potent regulators of tau aggregation as promising therapeutic targets.

neuroscience↗

bioRxiv2025

Sex differences in progressive multiple sclerosis brain gene expression in oligodendrocytes and OPCs

Multiple sclerosis is a neurological autoimmune disease with sex-imbalanced incidence; in the USA, the disease is more likely to effect females at a ratio of 3:1. In addition, males are more likely to have a more severe disease course at time of diagnosis. Questions about both causes and downstream effects of this disparity remain. We aim to investigate gene expression differences at a cellular level while considering sex to discover fine-scale sex disparities. These investigations could provide new avenues for treatment targeting, or treatment planning based on sex. Public single-nuclei RNA-sequencing data from three publications of progressive MS including control brains were analysed using the Seurat R package. Differential gene and pathway expression was looked at both within a specific data set which has sub-lesion level sample dissection and across all studies to provide a broader lens. This allowed for the consideration of cell types and spatial positioning in relation to the interrogated lesion in some of the calculations. Our analysis showed expression changes in the female MS oligodendrocytes and oligodendrocyte progenitor cells compared to healthy controls, which were not observed in the corresponding male affected cells. Differentially up-regulated genes in females include increased HLA-A in the oligodendrocytes, and increased clusterin in the oligodendrocyte progenitor cells. There are also several mitochondrial genes in both the oligodendrocytes and oligodendrocyte progenitors which are up-regulated in females, including several directly involved in electron transport and which have previously been associated with neurodegenerative diseases. These results point to altered states in oligodendrocyte progenitors and oligodendrocytes that in combination with known physiological dissimilarities between sexes may denote different programming in males and females in response to the onset of demyelinating lesions. The potential for increased debris clearance mediated by clusterin and availability of oligodendrocyte progenitors in females may indicate an environment more primed for repair, potentially including remyelination. This could contribute to the disparity in etiology in females versus males.

neuroscience↗

bioRxiv2025

Distinct memory CD4 subset tropism of two CCR5-tropic HIV-1 in a rapid progressor

CCR5 tropic HIV-1 mainly infect memory CD4+ T cells. Memory CD4+ T cells are heterogenous which comprise central memory (CM), transitional memory (TM) and effector memory (EM) subsets based on cell differentiation stages. Previous studies demonstrate that low viral burden in the CM subset correlates with long-term non-progressive HIV-1 infection (1, 2) and is a hallmark of non-pathogenetic SIV infection (3, 4). These findings indicate the importance of CD4 subset tropism of HIV/SIV in determining disease progression. However, an important, yet unanswered question is whether CCR5 HIV-1 variants have different memory CD4 subset preferences. Here, we demonstrate clear compartmentalization of two CCR5 HIV-1 in different memory CD4 subsets in vivo.

microbiology↗

bioRxiv2024

Design of an Artificial Natural Killer Cell Mimicking System to Target Tumour Cells

NK cell mimics are assemblies of a cell membrane and a template that replicate biomimetic features and physicochemical properties, respectively. For the reported design, we used the cell membrane from human NK cell (KHYG-1) line and gelatin microspheres as a template. The gelatin microspheres were reinforced via DMTMM cross-linking in a water-in-oil emulsion to exhibit tunable Youngs modulus. These engineered NK cell mimics were found to be non-toxic, non-inflammatory, and capable of evading macrophage detection when tested with differentiated THP-1 cells. In vitro studies showed significant interaction/proximity of the mimics with cancer cells when tested in 2D cultures of breast cancer cells (MDA-MB-231), 3D spheroids of liver (HepG2) and colon (HT-29) cancer cell models, and a zebrafish breast cancer xenograft (MDA-MB-231) model. The NK cell mimics also evaded macrophage detection in a Kdrl:EGFP Spil: Ds Red zebrafish model. In a pilot assessment, loading and release of the sialyltransferase inhibitor (STI, 3Fax-Peracetyl Neu5Ac) using NK cell mimics significantly reduced -2,6 sialylation in 2D cultures of MDA-MB-231 cells, demonstrating the STIs intact functionality in inhibiting sialylation. These findings collectively underscore the promising potential of engineered NK cell mimics as versatile tools in cancer research and therapeutic applications.

bioengineering↗

bioRxiv2024

Transcriptional analysis of efferocytosis in mouse skin wounds

Defects in apoptotic cell clearance, or efferocytosis, can cause inflammatory diseases and prevent tissue repair due in part to inducing a pro-repair transcriptional program in phagocytic cells like macrophages. While the cellular machinery and metabolic pathways involved in efferocytosis have been characterized, the precise efferocytic response of macrophages is dependent on the identity and macromolecular cues of apoptotic cells, and the complex tissue microenvironment in which efferocytosis occurs. Here, we find that macrophages undergoing active efferocytosis in mid-stage mouse skin wounds in vivo display a pro-repair gene program, while efferocytosis of apoptotic skin fibroblasts in vitro also induces an inflammatory transcription response. These data provide a resource for understanding how the skin wound environment influences macrophage efferocytosis and will be useful for future investigations that define the role of efferocytosis during tissue repair.

cell biology↗

bioRxiv2024

Microglia determine beta-amyloid plaque burden but are non-essential for downstream pathology

Evidence points to a role for microglia in Alzheimers disease (AD) risk, although their position in the pathological cascade is incompletely understood, prompting us to generate a model of {beta}-amyloidopathy lacking microglia. We find evidence that microglia promote plaque formation and creation of an A{beta} fibril-rich zone surrounding the plaque core. However, plaque-proximal reactive astrogliosis, synapse loss, and neurite dystrophy are still observed in the absence of microglia.

neuroscience↗

bioRxiv2024

The Scalable Variant Call Representation: Enabling Genetic Analysis Beyond One Million Genomes

The Variant Call Format (VCF) is widely used in genome sequencing but scales poorly. For instance, we estimate a 150,000 genome VCF would occupy 900 TiB, making it both costly and complicated to produce and analyze. The issue stems from VCFs requirement to densely represent both reference-genotypes and allele-indexed arrays. These requirements lead to unnecessary data duplication and, ultimately, very large files. To address these challenges, we introduce the Scalable Variant Call Representation (SVCR). This representation reduces file sizes by ensuring they scale linearly with samples. SVCR achieves this by adopting reference blocks from the Genomic Variant Call Format (GVCF) and employing local allele indices. SVCR is also lossless and mergeable, allowing for N+1 and N+K incremental joint-calling. We present two implementations of SVCR: SVCR-VCF, which encodes SVCR in VCF format, and VDS, which uses Hails native format. Our experiments confirm the linear scalability of SVCR-VCF and VDS, in contrast to the super-linear growth seen with standard VCF files. We also discuss the VDS Combiner, a scalable, open-source tool for producing a VDS from GVCFs and unique features of VDS which enable rapid data analysis. SVCR, and VDS in particular, ensure the scientific community can generate, analyze, and disseminate genetics datasets with millions of samples.

bioinformatics↗

bioRxiv2023

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naive and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naive, were productively infected. Increased CD4+ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

microbiology↗

bioRxiv2023

CHARR efficiently estimates contamination from DNA sequencing data

DNA sample contamination is a major issue in clinical and research applications of whole genome and exome sequencing. Even modest levels of contamination can substantially affect the overall quality of variant calls and lead to widespread genotyping errors. Currently, popular tools for estimating the contamination level use short-read data (BAM/CRAM files), which are expensive to store and manipulate and often not retained or shared widely. We propose a new metric to estimate DNA sample contamination from variant-level whole genome and exome sequence data, CHARR, Contamination from Homozygous Alternate Reference Reads, which leverages the infiltration of reference reads within homozygous alternate variant calls. CHARR uses a small proportion of variant-level genotype information and thus can be computed from single-sample gVCFs or callsets in VCF or BCF formats, as well as efficiently stored variant calls in Hail VDS format. Our results demonstrate that CHARR accurately recapitulates results from existing tools with substantially reduced costs, improving the accuracy and efficiency of downstream analyses of ultra-large whole genome and exome sequencing datasets.

bioinformatics↗

bioRxiv2023

Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

Intratumoral B cell responses are associated with more favorable clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). However, the antigens driving these B cell responses are largely unknown. We sought to discover these antigens by using single-cell RNA sequencing (scRNA-Seq) and immunoglobulin (Ig) sequencing of tumor-infiltrating immune cells from seven primary PDAC samples. We identified activated T and B cell responses and evidence of germinal center reactions. Ig sequencing identified plasma cell (PC) clones expressing isotype-switched and hyper-mutated Igs, suggesting the occurrence of T cell-dependent B cell responses. We assessed the reactivity of 41 recombinant antibodies that represented the products of 235 PCs and 12 B cells toward multiple cell lines and PDAC tissues, and observed frequent staining of intracellular self-antigens. Three of these antigens were identified: the filamentous actin (F-actin), the nucleic protein, RUVBL2, and the mitochondrial protein, HSPD1. Antibody titers to F-actin and HSPD1 were elevated in the plasma of PDAC patients, and also detectable in healthy donors. Thus, PCs in PDAC produce auto-antibodies reacting with intracellular self-antigens, which may result from promotion of pre-existing, autoreactive B cell responses. These observations indicate that the chronic inflammatory microenvironment of PDAC can support the adaptive immune response.

cancer biology↗

bioRxiv2023

Transcription elongation defects link oncogenic splicing factor mutations to targetable alterations in chromatin landscape

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two splicing factors commonly mutated in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy. HIGHLIGHTS- Oncogenic mutations in SF3B1 and U2AF1 cause a gene body RNAPII transcription elongation defect - The elongation defect is linked to impaired assembly of early spliceosome complexes and leads to replication stress and changes to chromatin landscape - RNAPII elongation defects in SF3B1K700E are normalized by modulating epigenetic factors but 3 cryptic splicing events are not reversed - Targeting the Sin3/HDAC pathway to normalize RNAPII elongation defect is a potential therapeutic approach in these disorders GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/530019v3_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@dc6d22org.highwire.dtl.DTLVardef@180f36borg.highwire.dtl.DTLVardef@ed341forg.highwire.dtl.DTLVardef@f8dfb2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Tracking coreceptor switch of the transmitted/founder HIV-1 identifies co-evolution of HIV-1 antigenicity, coreceptor usage and CD4 subset targeting

The CCR5 (R5) to CXCR4 (X4) coreceptor switch in natural HIV-1 infection is associated with faster progression to AIDS, but the underlying mechanisms remain unclear. The difficulty in capturing the earliest moment of coreceptor switch in vivo limits our understanding of this phenomenon. Here, by tracking the evolution of the transmitted/founder (T/F) HIV-1 in a prospective cohort of individuals at risk for HIV-1 infection identified very early in acute infection, we investigated this process with high resolution. The earliest X4 variants evolved from the R5 tropic T/F strains. Strong X4 usage can be conferred by a single mutation. The mutations responsible for coreceptor switch can confer escape to neutralization and drive X4 variants to replicate mainly in the central memory and naive CD4+ T cells. We propose a novel concept to explain the co-evolution of virus antigenicity and entry tropism termed "escape by shifting". This concept posits that for viruses with receptor or coreceptor flexibility, entry tropism alteration represents a mechanism of immune evasion in vivo.

microbiology↗

bioRxiv2023

Different Coexisting Mpox Lineages Were Continuously Circulating in Humans Prior to 2022

The origin and hazardous potential of human mpox is obscured by a lack of genomic data between the 2018, when exportations from Nigeria were recorded, and 2022 when the global outbreak started. Here, 18 genomes from patients across southern Nigeria in 2019/20 reveal multiple lineages of Monkeypox virus have achieved sustained human-to-human transmission, co-existing in humans for several years and accumulating mutations consistent with APOBEC3 activity suggesting the virus in humans is now segregated from its natural reservoir. Remarkably, three genomes have disruptions in the A46R gene, which contributes to innate immune modulation. The data demonstrates that the A.2 lineage, multiply exported to North America since 2021 independently of the global outbreak, has persisted in Nigeria for more than two years prior to its latest exportation. One-Sentence SummaryMpox is now a human diseae evolving in humans with multiple variants taking separate paths towards adaptation, some analogous to those of Variola

microbiology↗

bioRxiv2022

Reactivation of memory-encoding dentate gyrus neurons during memory consolidation is associated with subregion-specific, learning- and sleep-mediated biosynthetic changes

Post-learning sleep is essential for hippocampal memory processing, including contextual fear memory (CFM) consolidation. We used targeted recombination in activated populations (TRAP) to label context-encoding engram neurons in the hippocampal dentate gyrus (DG) and assessed reactivation of these neurons after fear learning. Post-learning sleep deprivation (SD), which impairs CFM consolidation, selectively disrupted reactivation of inferior blade DG engram neurons. This change was linked to more general suppression of neuronal activity markers in the inferior, but not superior, DG blade by SD. To further characterize how learning and subsequent sleep or SD affect these (and other) hippocampal subregions, we used subregion-specific spatial profiling of transcripts and proteins. We found that transcriptomic responses to sleep loss differed greatly between hippocampal regions CA1, CA3, and DG inferior blade, superior blade, and hilus - with activity-driven transcripts, and those associated with cytoskeletal remodeling, selectively suppressed in the inferior blade. Critically, learning-driven transcriptomic changes, measured 6 h following contextual fear learning, were limited to the two DG blades, differed dramatically between the blades, and were absent from all other regions. These changes suggested an increase in glutamatergic receptor signaling, and a decrease in GABA receptor signaling, during CFM consolidation. Protein abundance across hippocampal subregions was also differentially impacted by learning and sleep, with most alterations to protein expression restricted to DG. Together, these data suggest that the DG is critical for sleep-dependent memory consolidation, and that the effects of sleep loss on the hippocampus are highly subregion-specific, even within the DG itself.

neuroscience↗