Biology subjects

Lee, A.

Publications and source records attributed to Lee, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Inflammation induced Th17 cells synergize with the inflammation-trained microbiota to mediate host-resiliency against intestinal injury

Background and AimsInflammation can generate pathogenic Th17 cells and cause a inflammatory dysbiosis. In the context of Inflammatory Bowel Disease (IBD) these inflammatory Th17 cells and dysbiotic microbiota may perpetuate injury to intestinal epithelial cells (IECs). However, many models of IBD like T-cell transfer colitis and IL-10-/- mice rely on the absence of regulatory pathways, so it is difficult to tell if inflammationcan also induce protective Th17 cells. MethodsWe subjected C57BL6, RAG1-/- or JH-/- mice to systemic or gastrointestinal (GI) Citrobacter rodentium (Cr). Mice were then subject to 2.5% dextran sodium sulfate to cause epithelial injury. Fecal microbiota transfer was performed by bedding transfer and co-housing. Flow cytometry, qPCR, 16s sequencing and histology were used to assess parameters. ResultsTransient inflammation with GI but not systemic Cr was protective from subsequent intestinal injury. This was replicated with sequential DSS collectively indicating that transient inflammation provides tissue-specific protection. Inflammatory Th17 cells that have a tissue resident memory signature expanded in the intestine. Experiments with reconstituted RAG1-/-, JH-/- mice and cell trafficking inhibitors showed that inflammation induced Th17 cells were required for protection. Fecal microbiota transfer showed that the inflammation-trained microbiota was necessary for protection, likely by maintaining protective Th17 cells in situ. ConclusionInflammation can generate protective Th17 cells which synergize with the inflammation-trained microbiota to provide host resiliency against subsequent injury, indicating that inflammation induced Th17 tissue resident memory T cells are heterogenous and contain protective subsets.

immunology↗

bioRxiv2024

Have AI-Generated Texts from LLM Infiltrated the Realm of Scientific Writing? A Large-Scale Analysis of Preprint Platforms

Since the release of ChatGPT in 2022, AI-generated texts have inevitably permeated various types of writing, sparking debates about the quality and quantity of content produced by such large language models (LLM). This study investigates a critical question: Have AI-generated texts from LLM infiltrated the realm of scientific writing, and if so, to what extent and in what setting? By analyzing a dataset comprised of preprint manuscripts uploaded to arXiv, bioRxiv, and medRxiv over the past two years, we confirmed and quantified the widespread influence of AI-generated texts in scientific publications using the latest LLM-text detection technique, the Binoculars LLM-detector. Further analyses with this tool reveal that: (1) the AI influence correlates with the trend of ChatGPT web searches; (2) it is widespread across many scientific domains but exhibits distinct impacts within them (highest: computer science, engineering sciences); (3) the influence varies with authors who have different language speaking backgrounds and geographic regions according to the location of their affiliations (Italy, China, etc.); (4) AI-generated texts are used in various content types in manuscripts (most significant: hypothesis formulation, conclusion summarization); (5) AI usage has a positive influence on papers impact, measured by its citation numbers. Based on these findings, suggestions about the advantages and regulation of AI-augmented scientific writing are discussed.

scientific communication and education↗

bioRxiv2024

Misspellings or miscellings- non-verifiable cell lines in cancer research publications

Reproducible laboratory research relies on correctly identified reagents. We have previously described human gene research papers with wrongly identified nucleotide sequence reagent(s), including papers studying miR-145. Manually verifying reagent identities in more recent miR-145 papers found 20/36 (56%) and 6/36 (17%) miR-145 papers with misidentified nucleotide sequence reagent(s) and human cell line(s), respectively. We also found 5 cell line identifiers in two miR-145 papers with wrongly identified nucleotide sequences and cell lines, and 18 identifiers published elsewhere that did not correspond to indexed cell lines. These cell line identifiers were described as non-verifiable, as their identities appeared uncertain. Studying 420 papers that mentioned 8 different non-verifiable cell line identifier(s) found 235 papers (56%) that appeared to refer to BGC-803, BSG-803, BSG-823, GSE-1, HGC-7901, HGC-803 and/or MGC-823 as independent cell lines. We could not find publications describing how these cell lines were established, and they were not indexed in claimed externally accessible cell line repositories. While some papers stated that STR profiles had been generated for BGC-803, GSE-1 and/or MGC-823 cells, no STR profiles were identified. In summary, non-verifiable human cell lines represent new challenges to research reproducibility and require further investigation to clarify their identities. Novelty and Impact StatementThrough verifying reagent identities in research publications, our team found 23 non-verifiable human cell line identifiers, most of which could represent misspellings of contaminated cancer cell lines. Of 8 identifiers studied in detail, 7 non-verifiable identifiers were unexpectedly referred to as independent cell lines across 235 publications. We therefore describe a process "miscelling", where published cell lines lack descriptions of how they were established, cannot be found in claimed external repositories and lack STR profiles.

cancer biology↗

bioRxiv2024

MU-BRAIN: MUltiethnic Brain Rna-seq for Alzheimer INitiative

Alzheimers Disease (AD) exhibits a complex molecular and phenotypic profile. Investigating gene expression plays a crucial role in unraveling the diseases etiology and progression. Transcriptome data across ethnic groups lack, negatively impacting equity in intervention and research. We employed 565 brains across six U.S. brain banks (n=399 non-Hispanic Whites, n=113 Hispanics, n=12 African Americans) to generated bulk RNA sequencing from prefrontal cortex. We sought to identify cross-ancestry and ancestry-specific differentially expressed genes (DEG) across Braak stages, adjusting for sex, age at death, and RNA quality metrics. We further validated our findings using the Religious Orders Study/Memory Aging Project brains (ROS/MAP; n=1,095) and performed metanalysis (n=1,660). We conducted Gene Set and Variation and Enrichment analysis (GSVA; GSEA). We employed a machine-learning approach for phenotype prediction and gene prioritization to construct a polytranscriptomics risk score (PTRS) splitting our sample into training and testing sub-samples, either randomly or by ethnicity ("ancestry-agnostic" and "ancestry-aware", respectively). Lastly, we validated top DEG using single-nucleus RNA sequencing (snRNAseq) data. We identified several DEG associated with Braak staging: AD-known genes VGF (Padj =3.78E- 07) and ADAMTS2 (Pad j=1.21E-04) were consistently differentially expressed across statistical models, ethnicities, and replicated in ROS/MAP. Genes from the heat shock protein (HSP) family, e.g. HSPB7 (Padj =3.78E-07), were the top differentially expressed genes and replicated in ROS/MAP. Ethnic-stratified analyses prioritized TNFSF14 and SPOCD1 as top Hispanics DEG. GSEA highlighted "Alzheimer disease" (Padj =4.24E-06) and "TYROBP causal network in microglia" (Padj =1.68E-08) pathways. Up- and down-regulated genes were enriched in several pathways (e.g. "Immune response activation signal pathways", "Vesicle-mediated transport in synapse", "cognition"). Ancestry-agnostic and ancestry-aware PTRS effectively classified brains (AUC=0.77 and 0.73 respectively) and replicated in ROS/MAP. snRNAseq validated prioritized genes, including VGF (downregulated in neurons; Padj=1.1 E-07). This is the largest diverse AD transcriptome in post-mortem brain tissue, to our knowledge. We identified perturbated genes, pathways and network expressions in AD brains resulting in cross- ethnic and ethnic-specific findings, ultimately highlighting the diversity within AD pathogenesis. The latter underscores the need for an integrative and personalized approach in AD studies.

genetics↗

bioRxiv2024

Identification of early-stage liver fibrosis by modifications in the interstitial space diffusive microenvironment using fluorescent single-walled carbon nanotubes

During liver fibrosis, recurrent hepatic injuries lead to accumulation of collagen and other extracellular matrix components in the interstitial space, ultimately disrupting liver functions. Early stages of liver fibrosis may be reversible, but opportunities for diagnosis at these stages are currently limited. Here, we show that the alterations of the interstitial space associated with fibrosis can be probed by tracking individual fluorescent single-walled carbon nanotubes (SWCNTs) diffusing in that space. In a mouse model of early liver fibrosis, we find that nanotubes generally explore elongated areas, whose length decrease as the disease progresses even in regions where histopathological examination does not reveal fibrosis yet. Furthermore, this decrease in nanotube mobility is a purely geometrical effect, as the instantaneous nanotube diffusivity stays unmodified. This work establishes the promise of SWCNTs both for the diagnosis of liver fibrosis at an early stage, and for more in-depth studies of the biophysical effects of the disease.

cancer biology↗

bioRxiv2023

A non-conducting role of the Cav1.4 Ca2+ channel drives homeostatic plasticity at the cone photoreceptor synapse

In congenital stationary night blindness type 2 (CSNB2)--a disorder involving the Cav1.4 (L-type) Ca2+ channel--visual impairment is mild considering that Cav1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Cav1.4. Surprisingly, Cav3 (T-type) Ca2+ currents were detected in cones of G369i KI mice and Cav1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Cav1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Cav1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Cav1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.

neuroscience↗

bioRxiv2023

Inferring therapeutic vulnerability within tumors through integration of pan-cancer cell line and single-cell transcriptomic profiles

Single-cell RNA sequencing greatly advanced our understanding of intratumoral heterogeneity through identifying tumor subpopulations with distinct biologies. However, translating biological differences into treatment strategies is challenging, as we still lack tools to facilitate efficient drug discovery that tackles heterogeneous tumors. One key component of such approaches tackles accurate prediction of drug response at the single-cell level to offer therapeutic options to specific cell subpopulations. Here, we present a transparent computational framework (nicknamed scIDUC) to predict therapeutic efficacies on an individual-cell basis by integrating single-cell transcriptomic profiles with large, data-rich pan-cancer cell line screening datasets. Our method achieves high accuracy, with predicted sensitivities easily able to separate cells into their true cellular drug resistance status as measured by effect size (Cohens d > 1.0). More importantly, we examine our methods utility with three distinct prospective tests covering different diseases (rhabdomyosarcoma, pancreatic ductal adenocarcinoma, and castration-resistant prostate cancer), and in each our predicted results are accurate and mirrored biological expectations. In the first two, we identified drugs for cell subpopulations that are resistant to standard-of-care (SOC) therapies due to intrinsic resistance or effects of tumor microenvironments. Our results showed high consistency with experimental findings from the original studies. In the third test, we generated SOC therapy resistant cell lines, used scIDUC to identify efficacious drugs for the resistant line, and validated the predictions with in-vitro experiments. Together, scIDUC quickly translates scRNA-seq data into drug response for individual cells, displaying the potential as a first-line tool for nuanced and heterogeneity-aware drug discovery.

bioinformatics↗

bioRxiv2023

Neutrophil-derived IL-17 limits protective host responses and promotes tuberculosis pathogenesis

The protective correlates of Mycobacterium tuberculosis (Mtb) infection-elicited host immune responses are incompletely understood. Here, we report pro-pathogenic crosstalk involving Ly6G+granulocytes (Ly6G+Gra), IL-17 and COX2. We show that in the lungs of Mtb-infected wildtype mice, either BCG-vaccinated or not, most intracellular bacilli are Ly6G+Gra-resident four weeks post-infection onwards. In the genetically susceptible IFN{psi} -/- mice, excessive Ly6G+Gra infiltration correlates with severe bacteraemia. Neutralizing IL-17 (anti-IL17mAb) and COX2 inhibition by celecoxib reverse Ly6G+Gra infiltration, associated pathology and death in IFN{psi} -/- mice. Surprisingly, Ly6G+Gra also serves as the major source of IL-17 in the lungs of Mtb-infected WT or IFN{psi} -/- mice. The IL-17-COX2-Ly6G+Gra interplay also operates in WT mice. Inhibiting ROR{psi}t, the key transcription factor for IL-17 production or COX2, reduces the bacterial burden in Ly6G+Gra, leading to reduced bacterial burden and pathology in the lungs of WT mice. In the Mtb-infected WT mice, COX2 inhibition abrogates IL-17 levels in the lung homogenates and significantly enhances BCGs protective efficacy, mainly by targeting the Ly6G+Gra-resident Mtb pool, a phenotype also observed when IL-17 is blocked by ROR{psi}t inhibitor. Furthermore, in pulmonary TB patients, high neutrophil count and IL-17 correlated with adverse treatment outcomes. Together, our results suggest that IL-17 and PGE2 are the negative correlates of protection, and we propose targeting the pro-pathogenic IL-17-COX2-Ly6G+Gra axis for TB prevention and therapy.

immunology↗

bioRxiv2023

MFSD7c functions as a transporter of choline at the blood-brain barrier

Mutations of MFSD7c (also known as Flvcr2), which is an orphan transporter, are linked to Fowler syndrome 1, 2. Here, we use Mfsd7c knockout mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics and detected differential changes of metabolites in the brains and livers of Mfsd7c knockout (Mfsd7c-/-) embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the levels of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch-clamp showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c is conserved in vertebrates, but not in yeasts. We show that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. Employing our transport assays, we showed that several missense mutations of human MFSD7c from Fowler patients had abolished or reduced choline transport activity. Mice lacking Mfsd7c in the CNS endothelial cells suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c is required for exporting choline derived from lysophosphatidylcholine (LPC) in the brain. Collectively, our work identifies MFSD7c as a choline transporter at the BBB. This study suggests that defective export of choline in the brain may be a cause of Fowler syndrome.

biochemistry↗

bioRxiv2023

A 4D transcriptomic map for the evolution of multiple sclerosis-like lesions in the marmoset brain

Single-time-point histopathological studies on postmortem multiple sclerosis (MS) tissue fail to capture lesion evolution dynamics, posing challenges for therapy development targeting development and repair of focal inflammatory demyelination. To close this gap, we studied experimental autoimmune encephalitis (EAE) in the common marmoset, the most faithful animal model of these processes. Using MRI-informed RNA profiling, we analyzed [~]600,000 single-nucleus and [~]55,000 spatial transcriptomes, comparing them against EAE inoculation status, longitudinal radiological signals, and histopathological features. We categorized 5 groups of microenvironments pertinent to neural function, immune and glial responses, tissue destruction and repair, and regulatory network at brain borders. Exploring perilesional microenvironment diversity, we uncovered central roles of EAE-associated astrocytes, oligodendrocyte precursor cells, and ependyma in lesion formation and resolution. We pinpointed imaging and molecular features capturing the pathological trajectory of WM, offering potential for assessing treatment outcomes using marmoset as a platform. One sentence summaryA cross-modality study to identify the spatiotemporal-based diversity of primate brain cells during white matter inflammatory demyelination to inform lesion detection, stratification, and management in multiple sclerosis.

neuroscience↗

bioRxiv2023

A brown fat-enriched adipokine, ASRA, is a leptin receptor antagonist that stimulates appetite

The endocrine control of food intake remains incompletely understood, and whether the leptin receptor (LepR)-mediated anorexigenic pathway in the hypothalamus is negatively regulated by a humoral factor is unknown. Here, we identify an appetite-stimulating factor - ASRA - that represents a peripheral signal of energy deficit and orthosterically antagonizes LepR signaling. Asra encodes an 8 kD protein that is abundantly and selectively expressed in adipose tissue and to a lesser extent, in liver. ASRA associates with autophagy vesicles and its secretion is enhanced by energy deficiency. In vivo, fasting and cold stimulate Asra expression and increase its protein concentration in cerebrospinal fluid. Asra overexpression attenuates LepR signaling, leading to elevated blood glucose and development of severe hyperphagic obesity. Conversely, either adipose- or liver-specific Asra knockout mice display increased leptin sensitivity, improved glucose homeostasis, reduced food intake, resistance to high-fat diet-induced obesity, and blunted cold-evoked feeding response. Mechanistically, ASRA acts as a high affinity antagonist of LepR. AlphaFold2-multimer prediction and mutational studies suggest that a core segment of ASRA binds to the immunoglobin-like domain of LepR, similar to the site 3 recognition of the A-B loop of leptin. While administration of recombinant wild-type ASRA protein promotes food intake and increases blood glucose in a LepR signaling-dependent manner, point mutation within ASRA that disrupts LepR-binding results in a loss of these effects. Our studies reveal a previously unknown endocrine mechanism in appetite regulation and have important implications for our understanding of leptin resistance.

molecular biology↗

bioRxiv2023

Genomic DNA extraction optimization and validation for genome sequencing using the marine gastropod Kellet's whelk

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellets whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. We identified an optimized protocol that balances efficiency, cost, and DNA extraction quality; the Salting out protocol. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellets whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina Novaseq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research efforts on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies.

genomics↗

bioRxiv2023

Integrated Organ Immunity: Antigen-specific CD4-T cell-derived IFN-γ induced by BCG imprints prolonged lung innate resistance against respiratory viruses

Bacille Calmette-Guerin (BCG) vaccination can confer non-specific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. Here, we show that mice immunized intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with SARS-CoV-2 and influenza. Protection was first evident between 14 - 21 days post vaccination, and lasted for at least 42 days. Remarkably, BCG induced a biphasic innate response in the lung, initially at day 1 and a subsequent prolonged phase starting at [~]15 days post vaccination, and robust antigen-specific Th1 responses. MyD88-dependent TLR signaling was essential for the induction of the innate and Th1 responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or IFN-{gamma} activity prior to infection obliterated innate activation and protection. Single cell and spatial transcriptomics revealed CD4-dependent expression of interferon-stimulated genes (ISGs) in myeloid, type II alveolar and lung epithelial cells. Thus, BCG elicits "integrated organ immunity" where CD4+ T cells act on local myeloid and epithelial cells to imprint prolonged antiviral innate resistance.

immunology↗

bioRxiv2023

Revealing abrupt transitions from goal-directed to habitual behavior

A fundamental tenet of animal behavior is that decision-making involves multiple controllers. Initially, behavior is goal-directed, driven by desired outcomes, shifting later to habitual control, where cues trigger actions independent of motivational state. Clark Hulls question from 1943 still resonates today: "Is this transition abrupt, or is it gradual and progressive?"1 Despite a century-long belief in gradual transitions, this question remains unanswered2,3 as current methods cannot disambiguate goal-directed versus habitual control in real-time. Here, we introduce a novel volitional engagement approach, motivating animals by palatability rather than biological need. Offering less palatable water in the home cage4,5 reduced motivation to work for plain water in an auditory discrimination task when compared to water-restricted animals. Using quantitative behavior and computational modeling6, we found that palatability-driven animals learned to discriminate as quickly as water-restricted animals but exhibited state-like fluctuations when responding to the reward-predicting cue--reflecting goal-directed behavior. These fluctuations spontaneously and abruptly ceased after thousands of trials, with animals now always responding to the reward-predicting cue. In line with habitual control, post-transition behavior displayed motor automaticity, decreased error sensitivity (assessed via pupillary responses), and insensitivity to outcome devaluation. Bilateral lesions of the habit-related dorsolateral striatum7 blocked transitions to habitual behavior. Thus, volitional engagement reveals spontaneous and abrupt transitions from goal-directed to habitual behavior, suggesting the involvement of a higher-level process that arbitrates between the two.

neuroscience↗

bioRxiv2023

A transient protein folding response targets aggregation in the early phase of TDP-43-mediated disease

Understanding the mechanisms that drive TDP-43 pathology is integral to combating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To address this, we sought to determine the timeline of proteomic alterations across disease course in TDP-43 proteinopathy. Using longitudinal quantitative proteomics analysis of cortex samples from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, we identified several distinct protein subsets characterized by temporal alterations in protein abundance across diverse biological pathways, including protein folding, intracellular transport, myelination, and neuronal synaptic function. Remarkably, neurons in the rNLS8 cortex elicited a transitory response primarily comprising protein-folding factors prior to and in the earliest stages of disease progression. This response included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, and proof-of-concept studies showed that DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures. Conversely, knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in the brains and spinal cords of mice. Lastly, the late disease proteomic signatures of rNLS8 mouse cortex strongly correlated with changes in human autopsy-derived TDP-43 proteinopathy tissues, indicating commonality of disease processes. Together, these findings reveal molecular mechanisms that regulate protein levels through distinct stages of ALS and FTLD progression, and suggest that protein folding factors that combat cytoplasmic TDP-43 protein aggregation could be protective in disease. HighlightsO_LIThe first longitudinal map of the cortex proteome throughout TDP-43-driven disease in a mouse model of cytoplasmic TDP-43 proteinopathy (rNLS8 mice). C_LIO_LICytoplasmic TDP-43 accumulation drives many dynamic changes to the cortex proteome, including increases in protein folding factors prior to disease onset. C_LIO_LIThe protein folding factor DNAJB5 decreases TDP-43 aggregation in HEK293 cells and primary cortical neurons and Dnajb5 knockout exacerbates cytoplasmic TDP-43-associated motor impairments in vivo. C_LIO_LIThe proteomic signature of the rNLS8 mouse cortex correlates strongly with postmortem brain tissue from human TDP-43 proteinopathies. C_LIO_LIA new webtool, TDP-map (https://shiny.rcc.uq.edu.au/TDP-map/), allows comparison of transcriptomic and proteomic datasets from mouse and human TDP-43 proteinopathy. C_LI

neuroscience↗

bioRxiv2023

Functional interrogation of twenty type 2 diabetesassociated genes using isogenic hESC-derived β-like cells

Genetic studies have identified numerous loci associated with type 2 diabetes (T2D), but the functional role of many loci has remained unexplored. In this study, we engineered isogenic knockout human embryonic stem cell (hESC) lines for 20 genes associated with T2D risk. We systematically examined {beta}-cell differentiation, insulin production and secretion, and survival. We performed RNA-seq and ATAC-seq on hESC-{beta} cells from each knockout line. Analyses of T2D GWAS signals overlapping with HNF4A-dependent ATAC peaks identified a specific SNP as a likely causal variant. In addition, we performed integrative association analyses and identified four genes (CP, RNASE1, PCSK1N and GSTA2) associated with insulin production, and two genes (TAGLN3 and DHRS2) associated with sensitivity to lipotoxicity. Finally, we leveraged deep ATAC-seq read coverage to assess allele-specific imbalance at variants heterozygous in the parental hESC line, to identify a single likely functional variant at each of 23 T2D GWAS signals.

genetics↗

bioRxiv2023

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. nELISA can measure both protein concentration and their post-translational modifications. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity nor impact on performance vs 1-plex signals, with sensitivities as low as 0.1 pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale inflammatory-secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating [~]1.4M protein data points in under a week; a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We validate nELISA for phenotypic screening, where its capacity to faithfully report hundreds of proteins make it a powerful tool across multiple stages of drug discovery.

bioengineering↗

bioRxiv2023

FRESH™ 3D bioprinted cardiac tissue, a bioengineered platform for in-vitro toxicology and pharmacology

There is critical need for a predictive model of human cardiac physiology in the drug development process for assessment of compound toxicology and pharmacology. In vitro two-dimensional monolayer culture of cardiomyocytes provides biochemical and cellular readouts, and in vivo small and large animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to an incomplete recapitulation of adult human cardiovascular physiology, which results in more difficult safety interpretations. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in a three-dimensional tissue structure. Here we advance this paradigm by implementing FRESH 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its adrenergic agonist drug response. To do this, we combined hiPSC-CMs, endothelial cells and fibroblasts in a cellular bioink and FRESH 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. Our results confirmed that FRESH 3D bioprinted cardiac tissues could be fabricated directly in a 24-well plate format, were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL, and within 14 days demonstrated reproducible calcium transients and fast conduction velocity of [~]25 cm/s. Interrogation of these cardiac tissues with the {beta}-adrenergic receptor agonist isoproterenol showed native-like positive chronotropic and inotropic responses, a combination of responses that is not typically observed in 2D monolayer models or standard 3D engineered heart tissue approaches. These results confirm that FRESH 3D bioprinted cardiac tissues represents a novel in vitro platform that enables early in vitro pharmacology and toxicology screening.

bioengineering↗