Biology subjects

Jain, M.

Publications and source records attributed to Jain, M..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

The nephronophthisis protein GLIS2/NPHP7 is required for the DNA damage response in kidney tubular epithelial cells

Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. While nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novel Nphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2Y122X). Homozygous mice develop cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibit an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys becomes apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with the in vivo data, the knockdown of Glis2 in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of GFP-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2Y122X and a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DNA damage response, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies. Author SummaryNephronophthisis (NPH) is a rare inherited kidney disease characterized by cyst formation, fibrosis, and kidney failure at a young age. It is typically caused by mutations in genes essential for the proper function of cilia, small sensory, antenna-like cell protrusions. However, emerging evidence suggests that genome instability and impaired DNA repair may also contribute to NPH. Thus, in some cases, ciliopathies may result from defects in nuclear proteins rather than ciliary proteins. To investigate this, we generated a mouse model with a defective NPHP7/Glis2 gene using genome-editing tools. The encoded protein GLIS2 has been described to be in the cilium and the nucleus, while its detailed function remained unclear. As these mice aged, they exhibited signs of DNA damage and later developed cystofibrotic kidney disease. Consistently, we demonstrated that the GLIS2 protein interacts with key DNA repair proteins and is rapidly recruited to sites of DNA damage and repair. Thus, inefficient DNA damage repair appears to contribute to kidney disease in this mouse model. These findings underscore the crucial role of genome stability in preventing kidney disease, providing new insights into the underlying causes of NPH.

cell biology↗

bioRxiv2024

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair for somatic benchmarks

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. In future publications, these data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

genomics↗

bioRxiv2024

Morphology of softening and translucency in the stems of tomato and eggplant seedlings due to infection by Ralstonia pseudosolanacearum F1C1

Ralstonia pseudosolanacearum F1C1 is a soil-borne phytopathogenic bacterium with a broad host range that infects several economically important crops. This study primarily focuses on the infection of this phytopathogen in two such important crop seedlings: tomato and eggplant. The observations of the study reveal a complex set of symptoms that include drooping and blackening of the seedling stem, as well as blackening, chlorosis, and curling of cotyledon leaves. Notably, the symptom of stem softening and translucency is seen primarily in the water-submerged stem regions of both root- and leaf-inoculated seedlings. While the wild-type R. pseudosolanacearum F1C1 strain and its weakly virulent mutant phcA::{Omega} exhibited this phenotype in both tomato and eggplant seedlings, the virulence-deficient hrpB::{Omega} mutant did so only in a few eggplant seedlings but not in tomato seedlings. By investigating these unique pathological phenotypes in the infected seedlings, this study uncovers a unique symptom previously not reported in seedling inoculation experiments. The work also touches upon the distinct escape mechanisms exhibited by seedlings, revealing how some of its host plants might resist wilting despite infection, offering new avenues for future research. These findings contribute to the understanding of R. pseudosolanacearum pathogenesis, shedding light on its virulence and host response mechanisms.

plant biology↗

bioRxiv2024

A Rigorous Multi-Laboratory Study of Known PDAC Biomarkers Identifies Increased Sensitivity and Specificity Over CA19-9 Alone

A blood test that enables surveillance for early-stage pancreatic ductal adenocarcinoma (PDAC) is an urgent need. Independent laboratories have reported PDAC biomarkers that could improve biomarker performance over CA19-9 alone, but the performance of the previously reported biomarkers in combination is not known. Therefore, we conducted a coordinated case/control study across multiple laboratories using common sets of blinded training and validation samples (132 and 295 plasma samples, respectively) from PDAC patients and non-PDAC control subjects representing conditions under which surveillance occurs. We analyzed the training set to identify candidate biomarker combination panels using biomarkers across laboratories, and we applied the fixed panels to the validation set. The panels identified in the training set, CA19-9 with CA199.STRA, LRG1, TIMP-1, TGM2, THSP2, ANG, and MUC16.STRA, achieved consistent performance in the validation set. The panel of CA19-9 with the glycan biomarker CA199.STRA improved sensitivity from 0.44 with 0.98 specificity for CA19-9 alone to 0.71 with 0.98 specificity (p < 0.001, 1000-fold bootstrap). Similarly, CA19-9 combined with the protein biomarker LRG1 and CA199.STRA improved specificity from 0.16 with 0.94 sensitivity for CA19-9 to 0.65 with 0.89 sensitivity (p < 0.001, 1000-fold bootstrap). We further validated significantly improved performance using biomarker panels that did not include CA19-9. This study establishes the effectiveness of a coordinated study of previously discovered biomarkers and identified panels of those biomarkers that significantly increased the sensitivity and specificity of early-stage PDAC detection in a rigorous validation trial.

cancer biology↗

bioRxiv2024

mRNA psi profiling using nanopore DRS reveals cell-type-specific pseudouridylation

Pseudouridine (psi) is one of the most abundant human mRNA modifications yet its functional impact on translation has remained unclear. Using direct RNA nanopore sequencing coupled with our Mod-p ID analytical framework, we mapped psi at single-nucleotide resolution across six immortalized human cell lines derived from diverse tissue types. Psi sites identified by nanopore sequencing were cross-validated using Illumina-based methods, confirming both positional accuracy and reproducibility. Unlike prior short-read approaches, nanopore sequencing provided the unique ability to quantify relative occupancy at each site and to detect multiple modifications on the same RNA molecule, revealing combinatorial modification patterns that cannot be captured otherwise. Integrating these psi maps with matched proteomic and ribosome profiling datasets, we find that psi modulates translation through two mechanistic modes: (i) single high-occupancy psi sites enhance translational efficiency and protein output, whereas (ii) clustered psi modifications promote ribosome pausing, decoupling translation efficiency from protein yield. This integrative, multi-omics framework provides a quantitative model of how psi stoichiometry and distribution along transcripts shape ribosome dynamics and proteome composition across human cell types.

genomics↗

bioRxiv2024

Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

The dopamine D1 receptor (D1R) has fundamental roles in voluntary movement and memory and is a validated drug target for neurodegenerative and neuropsychiatric disorders. However, previously developed D1R selective agonists possess a catechol moiety which displays poor pharmacokinetic properties. The first selective non-catechol D1R agonists were recently discovered and unexpectedly many of these ligands showed G protein biased signaling. Here, we investigate both catechol and non-catechol D1R agonists to validate potential biased signaling and examine if this impacts agonist-induced D1R endocytosis. We determined that most, but not all, non-catechol agonists display G protein biased signaling at the D1R and have reduced or absent {beta}-arrestin recruitment. A notable exception was compound (Cmpd) 19, a non-catechol agonist with full efficacy at both D1R-G protein or D1R-{beta}-arrestin pathways. In addition, the catechol ligand A-77636 was a highly potent, super agonist for D1R-{beta}-arrestin activity. When examined for agonist-induced D1R endocytosis, balanced agonists SKF-81297 and Cmpd 19 induced robust D1R endocytosis while the G protein biased agonists did not. The {beta}-arrestin super agonist, A-77636, showed significantly increased D1R endocytosis. Moreover, {beta}-arrestin recruitment efficacy of tested agonists strongly correlated with total D1R endocytosis. Taken together, these results indicate the degree of D1R signaling functional selectivity profoundly impacts D1R endocytosis regardless of pharmacophore. The range of functional selectivity of these D1R agonists will provide valuable tools to further investigate D1R signaling, trafficking and therapeutic potential. Significance StatementThe D1R is a validated therapeutic target and the recently discovered non-catechol D1R agonists have translational potential. We have systematically characterized several structurally distinct D1R agonists including non-catechols with balanced or G protein biased activity. When examined for agonist-induced D1R endocytosis, balanced agonists induced robust D1R endocytosis while G protein biased agonists did not. These results indicate the degree of D1R signaling functional selectivity profoundly impacts receptor endocytosis. This work also independently validates agonist tools to further investigate D1R activation in basic and translational research.

pharmacology and toxicology↗

bioRxiv2024

Probing enzyme-dependent pseudouridylation using direct RNA sequencing to assess neuronal epitranscriptome plasticity

Chemical modifications in mRNAs, such as pseudouridine (psi), can control gene expression. Yet, we know little about how they are regulated, especially in neurons. We applied nanopore direct RNA sequencing to investigate psi dynamics in SH-SY5Y cells in response to two perturbations that model a natural and unnatural cellular state: retinoic-acid-mediated differentiation (healthy) and exposure to the neurotoxicant, lead (unhealthy). We discovered that the expression of some psi writers change significantly in response to physiological conditions. We also found that globally, lead-treated cells have more psi sites but lower relative occupancy than untreated cells and differentiated cells. Interestingly, examples of highly plastic sites were accompanied by constant expression for psi writers, suggesting trans-regulation. Many positions were static throughout all three cellular states, suggestive of a "housekeeping" function. This study enables investigations into mechanisms that control psi modifications in neurons and its possible protective effects in response to cellular stress.

systems biology↗

bioRxiv2024

Comprehensive Invitro and Insilico Analysis of Secondary Metabolites from Datura metel: Promising Anti-Alzheimer's Therapeutics.

This research investigates secondary metabolites from Datura metel as potential anti-Alzheimers therapies. In vitro techniques isolated extracts for AD pathology targeting, with in silico analysis identifying gene targets for prevention. Apigenin, Luteolin, and Withanolide A were studied, each with 300 potential gene targets and core gene counts of 54, 52, and 58 respectively. Lipinskis Rule assessed their pharmacological properties, showing good absorption but limited blood-brain barrier penetration. Protein interaction mapping revealed shared targets among the compounds. GO enrichment and KEGG pathway analysis highlighted their impact on biological processes and pathways, suggesting their anti-Alzheimers potential. Luteolin notably reduced A{beta}1-42 levels by up to 35.2% (*p<0.05) in SH-SY5Y cells, positioning it and Withanolide A as promising multi-functional Alzheimers medications. These findings underscore the significance of Datura phytochemicals in AD prevention and treatment.

neuroscience↗

bioRxiv2024

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease. One Sentence SummaryPulmonary arterial hypertension pathophenotypes arise from allele-specific NCOA7 regulation of lysosome function and inflammatory oxysterol generation, as demonstrated by genomic and metabolomic association studies coupled with genetic and pharmacologic mechanistic evidence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/582142v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@896f6forg.highwire.dtl.DTLVardef@f31c2eorg.highwire.dtl.DTLVardef@bf46dcorg.highwire.dtl.DTLVardef@1c55686_HPS_FORMAT_FIGEXP M_FIG Graphical abstract. C_FIG

physiology↗

bioRxiv2024

Assessing methylation detection for primary human tissue using Nanopore sequencing

DNA methylation most commonly occurs as 5-methylcytosine (5-mC) in the human genome and has been associated with human diseases. Recent developments in single-molecule sequencing technologies (Oxford Nanopore Technologies (ONT) and Pacific Biosciences) have enabled readouts of long, native DNA molecules, including cytosine methylation. ONT recently upgraded their Nanopore sequencing chemistry and kits from R9 to the R10 version, which yielded increased accuracy and sequencing throughput. However the effects on methylation detection have not yet been documented. Here we performed a series of computational analyses to characterize differences in Nanopore-based 5mC detection between the ONT R9 and R10 chemistries. We compared 5mC calls in R9 and R10 for three human genome datasets: a cell line, a frontal cortex brain sample, and a blood sample. We performed an in-depth analysis on CpG islands and homopolymer regions, and documented high concordance for methylation detection among sequencing technologies. The strongest correlation was observed between Nanopore R10 and Illumina bisulfite technologies for cell line-derived datasets. Subtle differences in methylation datasets between technologies can impact analysis tools such as differential methylation calling software. Our findings show that comparisons can be drawn between methylation data from different Nanopore chemistries using guided hypotheses. This work will facilitate comparison among Nanopore data cohorts derived using different chemistries from large scale sequencing efforts, such as the NIH CARD Long Read Initiative.

genomics↗

bioRxiv2024

Pulmonary primary oxysterol and bile acid synthesis as a predictor of outcomesin pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH. One-Sentence SummaryThis work identifies pulmonary primary oxysterol and bile acid synthesis as altered in pulmonary arterial hypertension, thus establishing a new prognostic test for this disease.

systems biology↗

bioRxiv2023

AlphaFold2 structures template ligand discovery

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the {sigma}2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

pharmacology and toxicology↗

bioRxiv2023

Structure-Based Discovery of a NPFF1R Antagonist with Analgesic Activity

While opioid drugs remain among the most effective analgesics for pain management, adverse effects limit their use. Molecules that synergize with opioids, increasing analgesia without increasing side effects, could prove beneficial. A potential way to do so is via the RF-amide receptor system, as NPFFR1 agonists reduce {micro}- opioid receptor ({micro}OR)-based analgesia while antagonists increase it. These inferences are, however, clouded by the lack of selectivity of most NPFF1R ligands. Seeking selective antagonists of the NPFF1R, we screened a large virtual library against a homology model of NPFF1R. From 26 high-ranking molecules that were synthesized and tested, one antagonized NPFF1R with a Ki of 319 nM. Structure-based optimization led to a 22 nM antagonist of NPFF1R, compound 56, with selectivity against a large panel of GPCRs. When administered alone, 56 has no activity in mouse tail-flick nociception assays. However, coadministration of compound 56 and morphine produced significantly greater antinociception than did morphine alone, consistent with the notion that NPFF1R nociceptive activity occurs via modulation of {micro}OR signaling. Surprisingly, in the hot-plate assays 56 was analgesic by itself, suggesting that NPFF1R alone can also confer analgesia. At equi-analgesic doses, combinations of 56 with morphine reduced the common constipation side effect of morphine versus using morphine alone. The high selectivity of 56 and its activity in cooperation with morphine supports further analgesic development against NPFF1R and against the RF-amide family of receptors more generally.

pharmacology and toxicology↗

bioRxiv2023

Local read haplotagging enables accurate long-read small variant calling

Long-read sequencing technology has enabled variant detection in difficult-to-map regions of the genome and enabled rapid genetic diagnosis in clinical settings. Rapidly evolving third-generation sequencing platforms like Pacific Biosciences (PacBio) and Oxford nanopore technologies (ONT) are introducing newer platforms and data types. It has been demonstrated that variant calling methods based on deep neural networks can use local haplotyping information with long-reads to improve the genotyping accuracy. However, using local haplotype information creates an overhead as variant calling needs to be performed multiple times which ultimately makes it difficult to extend to new data types and platforms as they get introduced. In this work, we have developed a local haplotype approximate method that enables state-of-the-art variant calling performance with multiple sequencing platforms including PacBio Revio system, ONT R10.4 simplex and duplex data. This addition of local haplotype approximation makes DeepVariant a universal variant calling solution for long-read sequencing platforms.

bioinformatics↗

bioRxiv2023

CRISPR-COPIES: An in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and a human cell line. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals and support human gene and cell therapy applications. Graphical abstractOverview and application of CRISPR-COPIES in the field of biotechnology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/556564v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@c723a1org.highwire.dtl.DTLVardef@1f42594org.highwire.dtl.DTLVardef@1cf4bd6org.highwire.dtl.DTLVardef@7c1028_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19

As many as 10-30% of the over 760 million survivors of COVID-19 develop persistent symptoms, of which respiratory symptoms are among the most common. To understand the cellular and molecular basis for respiratory PASC, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry, single-cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples, and alveolar cytokine profiling in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed abnormalities involving 73% of the lung, which improved on subsequent imaging. Interstitial abnormalities suggestive of fibrosis on CT were associated with the increased numbers of neutrophils and presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid, reflecting unresolved epithelial injury. Persistent infection with SARS-CoV-2 was identified in six patients and secondary bacterial or viral infections in two others. These findings suggest that despite its heterogenous clinical presentations, respiratory PASC with radiographic abnormalities results from a common pathobiology characterized by the ongoing recruitment of neutrophils and profibrotic monocyte-derived alveolar macrophages driving lung fibrosis with implications for diagnosis and therapy.

immunology↗

bioRxiv2023

SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration

Reactive astrogliosis is a common pathological hallmark of central nervous system (CNS) injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce {beta}-amyloid plaques in mouse models of Parkinsons and Alzheimers disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Multicellular, IVT-derived, unmodified human transcriptome for nanopore direct RNA analysis

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analysis. We produced DRS datasets using modification-free transcripts from in vitro transcription (IVT) of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analysis of human transcripts.

genomics↗