Biology subjects

Shrestha, R.

Publications and source records attributed to Shrestha, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Structural basis for transcription activation through cooperative recruitment of MntR

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

biochemistry↗

bioRxiv2024

A prostate cancer gastrointestinal transcriptional phenotype may be associated with diminished response to AR-targeted therapy

BackgroundProstate cancer is a heterogenous disease, but once it becomes metastatic it eventually becomes treatment resistant. One mechanism of resistance to AR-targeting therapy is lineage plasticity, where the tumor undergoes a transformation to an AR-indifferent phenotype, most studied in the context of neuroendocrine prostate cancer (NEPC). However, activation of additional de- or trans-differentiation programs, including a gastrointestinal (GI) gene expression program, has been suggested as an alternative method of resistance. In this study, we explored the previously identified GI prostate cancer phenotype (PCa-GI) in a large cohort of metastatic castration-resistant prostate cancer (mCRPC) patient biopsy samples. MethodsWe analyzed a dataset of 634 mCRPC samples with batch effect corrected gene expression data from the West Coast Dream Team (WCDT), the East Coast Dream Team (ECDT), the Fred Hutchinson Cancer Research Center (FHCRC) and the Weill Cornell Medical center (WCM). Survival data was available from the WCDT and ECDT cohorts. We calculated a gene expression GI score using the sum of z-scores of genes from a published set of PCa-GI-defining genes (N=38). Survival analysis was performed using the Kaplan-Meier method and Cox proportional hazards regression with endpoint overall survival from time of biopsy to death of any cause. ResultsWe found that the PCa-GI score had a bimodal distribution, identifying a distinct set of tumors with an activated GI expression pattern. Approximately 35% of samples were classified as PCa-GI high, which was concordant with prior reports. Liver metastases had the highest median score but after excluding liver samples, 29% of the remaining samples were still classified as PCa-GI high, suggesting a distinct phenotype not exclusive to liver metastases. No correlation was observed between GI score and proliferation, AR signaling, or NEPC scores. Furthermore, the PCa-GI score was not associated with genomic alterations in AR, FOXA1, RB1, TP53 or PTEN. However, tumors with MYC amplifications showed significantly higher GI scores (p=0.0001). Patients with PCa-GI tumors had a shorter survival (HR=1.5 [1.1-2.1], p=0.02), but this result was not significant after adjusting for the liver as metastatic site (HR=1.2 [0.82-1.7], p=0.35). Patients with PCa-GI low samples had a better outcome after androgen receptor signaling inhibitors (ASI, abiraterone or enzalutamide) than other therapies (HR=0.37 [0.22-0.61], p=0.0001) while the benefit of ASI was smaller and non-significant for PCa-GI high samples (HR=0.55 [0.29-1.1], p=0.07). A differential pathway analysis identified FOXA2 signaling to be upregulated PCa-GI high tumors (FDR = 3.7 x 10-13). ConclusionsThe PCa-GI phenotype is prevalent in clinical mCRPC samples and may represent a distinct biological entity. PCa-GI tumors may respond less to ASI and could offer a strategy to study novel therapeutic targets.

cancer biology↗

bioRxiv2024

Differential TLR-ERK1/2 activity promotes viral ssRNA and dsRNA mimic-induced dysregulated immunity in macrophages

RNA virus induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as dysregulated immunity, is caused by viral single-stranded RNA (ssRNA) and double-stranded-RNA (dsRNA) mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We show that murine bone marrow-derived macrophages (BMDMs) stimulated with TLR3 and TLR7 agonists induce differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified a significantly higher cell-surface and endosomal TLR7 expression compared to TLR3, which further correlated with early and robust MAPK (pERK1/2 and p-P38) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2, p38, and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA-mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG responses, and blocking ERK1/2 activity would mitigate viral-RNA/TLR-induced dysregulated immunity.

immunology↗

bioRxiv2024

Impact of alternative splicing on Arabidopsis proteome

Limited proteomic evidence makes it unclear to what extent alternative splicing (AS) isoforms are translated and functionally relevant in eukaryotes. Here, we present a comprehensive proteomic analysis in plants using large-scale data mining, extensive fractionation of AspN-and trypsin-digested proteomes, and both label-free and TMT labeling. In total, we identified 471,196 peptides from 22,479 proteins by searching against Araport11, revealing 32,110 isoform-specific peptides. Using an integrated proteogenomic workflow coupled with SUPPA, we classified these peptides into 2,442 AS events, 879 of which involved intron retention (IR). Further analysis of unannotated events revealed 91 additional IRs that are translated, confirming that retained introns can give rise to peptides. AlphaFold modeling predicted the structural and functional impacts of these isoforms. Our dataset improved existing gene model annotations. By comparing wild-type plants with the AS mutant acinus pinin, we found that IR regulates transcript and protein abundance nonlinearly. Phenotypic assays confirmed the functional consequences, including reduced chlorophyll, impaired growth, and increased anthocyanin. Take together, our results provide large-scale proteomic evidence that AS isoforms are translated in plants, demonstrating how AS diversifies the proteome, regulates protein abundance, and affects growth and development.

plant biology↗

bioRxiv2024

The potential of undersown species identity vs. diversity to manage disease in crops

O_LIIn the absence of chemical control with its negative side effects, fungal pathogens can cause large yield losses, requiring us to develop agroecosystems that are inherently disease resistant. Grassland biodiversity experiments often find plant species diversity to reduce pathogen pressure, but whether incorporating high biodiversity levels in agricultural fields have similar effects remains largely unknown. C_LIO_LIWe tested if undersown plant species diversity could reduce barley disease, and whether the effect was mediated through above- or belowground mechanisms, by combining an agricultural field trial with a soil transplant experiment. C_LIO_LIAs predicted, barley disease decreased in the presence of undersown plants. Undersown species richness had no effect, but their abundance led to early season disease reduction. Aboveground mechanisms underpinned this disease reduction. Barley yield slightly decreased with increasing undersown species richness, and undersown species varied in their impact on yield. C_LIO_LIWe identified two undersown species with similar functional traits that contributed most to disease reduction and had the potential to increase barley yield. Furthermore, our results indicate that aboveground mechanisms caused this. We show that agroecosystem functioning can be improved without trade-offs on yield by targeted selection of undersown species. C_LI

ecology↗

bioRxiv2024

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration [≤] 50 {micro}M is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipes MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

molecular biology↗

bioRxiv2024

Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. In tumors, highly transcribed eccDNAs have been implicated in oncogenesis, neoantigen production and resistance to chemotherapy. Here we show that unstable microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures generate eccDNAs when integrated at a common ectopic site in human cells. These non-B DNA prone microsatellites form eccDNAs by replication-dependent mechanisms. The microsatellite-based eccDNAs are highly mutagenized and display template switches to sister chromatids and to nonallelic chromosomal sites. High frequency mutagenesis occurs within the eccDNA microsatellites and extends bidirectionally for several kilobases into flanking DNA and nonallelic DNA. Mutations include mismatches, short duplications, longer nontemplated insertions and large deletions. Template switching leads to recurrent deletions and recombination domains within the eccDNAs. Template switching events are microhomology-mediated, but do not occur at all potential sites of complementarity. Each microsatellite exhibits a distinct pattern of recombination, microhomology choice and base substitution signature. Depletion of Rad51, the COPS2 signalosome subunit or POL{eta} alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA breaks for the generation and circularization of mutagenized eccDNAs and genomic homologous recombination deficiency (HRD) scars.

molecular biology↗

bioRxiv2024

Mutation of highly conserved residues in loop 2 of the coronavirus macrodomain demonstrates that enhanced ADP-ribose binding is detrimental to infection

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. AUTHOR SUMMARYThe conserved CoV macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases by removing ADP-ribose from target proteins and is critical for CoV replication and pathogenesis. Mac1 is a potential therapeutic target for CoV disease and several groups are actively developing Mac1 inhibitors. However, we lack a basic knowledge of how many of the key residues in the Mac1 ADP-ribose binding pocket contribute to its biochemical and virological functions. In this study, we engineered alanine mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and recombinant viruses for both MERS-CoV and SARS-CoV-2 to determine their importance in both Mac1 biochemical functions and CoV infection. Interestingly, an isoleucine-to-alanine mutation in loop 2 of both MERS-CoV and SARS-CoV-2 Mac1 proteins enhanced ADP-ribose binding. But surprisingly, that proved to be detrimental to virus infection, indicating that this isoleucine functions to control Mac1 ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding to promote infection and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

microbiology↗

bioRxiv2023

Chloroplast Methyltransferase Homolog RMT2 is Involved in Photosystem I Biogenesis

Oxygen (O2), a dominant element in the atmosphere and essential for most life on Earth, is produced by the photosynthetic oxidation of water. However, metabolic activity can cause accumulation of reactive O2 species (ROS) and severe cell damage. To identify and characterize mechanisms enabling cells to cope with ROS, we performed a high-throughput O2 sensitivity screen on a genome-wide insertional mutant library of the unicellular alga Chlamydomonas reinhardtii. This screen led to identification of a gene encoding a protein designated Rubisco methyltransferase 2 (RMT2). Although homologous to methyltransferases, RMT2 has not been experimentally demonstrated to have methyltransferase activity. Furthermore, the rmt2 mutant was not compromised for Rubisco (first enzyme of Calvin-Benson Cycle) levels but did exhibit a marked decrease in accumulation/activity of photosystem I (PSI), which causes light sensitivity, with much less of an impact on other photosynthetic complexes. This mutant also shows increased accumulation of Ycf3 and Ycf4, proteins critical for PSI assembly. Rescue of the mutant phenotype with a wild-type (WT) copy of RMT2 fused to the mNeonGreen fluorophore indicates that the protein localizes to the chloroplast and appears to be enriched in/around the pyrenoid, an intrachloroplast compartment present in many algae that is packed with Rubisco and potentially hypoxic. These results indicate that RMT2 serves an important role in PSI biogenesis which, although still speculative, may be enriched around or within the pyrenoid. Significance StatementA high-throughput genetic screen was used to identify O2 sensitive mutants of Chlamydomonas reinhardtii (Chlamydomonas throughout) that experience elevated oxidative stress in the light relative to WT cells. Identification of genes altered in these mutants offers opportunities to discover activities that a) protect photosynthetic cells from oxidative damage, b) participate in rapid assembly of photosynthetic complexes, which would limit accessibility of intermediates to O2, and/or c) facilitate repair of damaged cellular complexes. A mutant from this screen disrupted for RMT2, originally described as encoding a Rubisco methyltransferase, was defective for PSI biogenesis. Additionally, RMT2 appears to be enriched in/around the pyrenoid, a chloroplast localized compartment harboring much of the Chlamydomonas Rubisco, raising the possibility that this compartment plays a role in PSI biogenesis.

plant biology↗

bioRxiv2023

SECRET AGENT O-GlcNAcylates hundreds of proteins involved in diverse cellular processes in Arabidopsis

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY (SPY), with SPY also exhibiting O-fucosylation activity. To investigate SECs role as a major O-GlcNAc transferase in plants, we utilized LWAC enrichment and SILIA labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating SECs critical role in mediating O-GlcNAcylation. Through a comprehensive approach, combining HCD and EThcD fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined EThcD fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

plant biology↗

bioRxiv2023

Inactivation of Highly Transmissible Livestock and Avian Viruses Including Influenza A and Newcastle Disease Virus for Molecular Diagnostics

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection but maintains the nucleic acid quality required for diagnostic PCR testing. This inactivation method is needed to alleviate concerns about transmission potential, reduce shipping complications and cost, and allow testing in lower containment laboratories to improve disease diagnostics by improving turn-around time. This study evaluated a panel of ten surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial (PrimeStore(R)) molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99% as determined by infectivity and serial passage assays. However, detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and shipment of infectious substances. Contribution to the fieldActive surveillance and confirmatory testing efforts are in place to protect animals in the United States from certain highly contagious diseases and to limit financial impacts to consumers and producers when the food supply is disrupted. Confirmatory testing typically utilizes nucleic acid detection to identify active infection. Testing is required to be completed in high containment facilities due to the elevated pathogenicity and impact potential of animal diseases. The requirement for testing in high containment facilities limits the ability for regional and state laboratories to test for Tier 1 select agents. Shipment of diagnostic samples is costly, as well as time and temperature sensitive to avoid deterioration of sample quality needed for testing. These constraints lengthen response time and testing turn-around time. Here, we showed that a commercial (PrimeStore(R)) molecular transport media (PSMTM) completely inactivated all viruses tested without affecting nucleic acid detection/integrity. These data suggest that highly contagious agents are effectively inactivated by PSMTM without compromising the nucleic acid needed for diagnostic testing. These data provide support that this inactivation method can be utilized during sample collection to reduce constraints in disease diagnostics and in reagent sharing among international laboratories.

microbiology↗

bioRxiv2023

Cell of origin epigenetic priming determines susceptibility to Tet2 mutation

Hematopoietic stem cell mutations can result in clonal hematopoiesis (CH) but the clinical outcomes are heterogeneous. The nature of the founder mutation and secondary mutations likely drive emergent neoplastic disease. We investigated how the state of the cell of origin where the Tet2 mutation occurs affects susceptibility to that commonly occurring CH mutation. Here, we provide evidence that risk is written in the epigenome of the cell of origin. By characterizing cell states that underlie myeloid differentiation and linking this information to an inducible system to assess myeloid progenitor clones, we provide evidence that epigenetic markers of the cell where Tet2 mutation occurs stratifies clonal behaviors. Specifically, Sox4 fosters a global cell state of high sensitization towards Tet2 KO. Using GMP and primary HSC models, we show that Sox4 promotes cell dedifferentiation, alters cell metabolism and increases the in vivo clonal output of mutant cells. Our results validate the hypothesis that epigenetic features can predispose specific clones for dominance and explain why an identical mutation can result in different outcomes.

cancer biology↗

bioRxiv2023

A novel phenylpropanoid methyl esterase enables catabolism of aromatic compounds that inhibit biological nitrification

Agriculture is the largest source of greenhouse gases (GHG) production. Conversion of nitrogen fertilizers into more reduced forms by microbes through a process known as biological nitrification drives GHG production, enhances proliferation of toxic algal blooms, and increases cost of crop production. Some plants reduce biological nitrification in soils by exuding a diverse array of biological nitrification inhibitors (BNIs) that inhibit the ammonium oxidizing microbes responsible for nitrification. Applying synthetic biology to enhance and transfer BNI production into food and bioenergy crops is a promising approach to reduce nitrification but the success of this strategy is contingent upon improving our limited understanding of BNIs mechanisms-of-action and degradation in the soil. We addressed this gap by investigating the previously unknown metabolic route through which a prominent class of aromatic BNIs known as phenylpropanoid methyl esters (PPMEs) are catabolized. While neither transcriptomics (RNAseq) or high-throughput functional genomics (RB-TnSeq) methods reduced the genetic search space for pathway discovery into a tenable number of genes, the combination of narrowed the search space to a collection of 4 proteins of unknown function. Using genetic and biochemical analyses we found that two previously uncharacterized enzymes, including a novel phenylpropanoid methyl esterase, funnel PPMEs into an established phenylpropanoid catabolism pathway. Transfer of these two enzymes into bacteria capable of using other phenylpropanoids use of PPMEs as carbon sources. This work both provided insight into BNI catabolism and is the first step towards development of model in vivo plant-microbe systems for studying BNI mechanisms under well controlled conditions.

microbiology↗

bioRxiv2023

Sorgoleone degradation by sorghum-associated bacteria; an opportunity for enforcing plant growth promotion

Metabolite exchange between plant roots and their associated rhizosphere microbiomes underpins plant growth promotion by microbes. Sorghum bicolor is a cereal crop that feeds animals and humans and is used for bioethanol production. Its root tips exude large amounts of a lipophilic benzoquinone called sorgoleone. Sorgoleone is an allelochemical that suppresses the growth of competing plant seedlings and is mineralized by microbes in soil. As an avenue to understand how sorghum and its root microbiome may be connected through root exudates, we identified the molecular determinants of microbial sorgoleone degradation and the distribution of this trait among microbes. We isolated and studied from sorghum-associated soils, three bacterial strains classified as Acinetobacter, Burkholderia, and Pseudomonas species that grow with sorgoleone as a sole carbon and energy source. The genomes of these strains were sequenced and subjected to transcriptomic and gene fitness analyses to identify candidate sorgoleone degradation genes. Follow up mutational analysis showed that sorgoleone catabolism is dependent on four contiguous genes that are conserved among the species we sequenced. Phylogenetic analysis of the sorgoleone degradation gene cluster showed that sorgoleone catabolism is enriched in sorghum-associated Streptomyces strains. The discovery of bacteria that grow on a compound like sorgoleone that is plant specific and not widely distributed in the environment, provides an opportunity to study how a plant exudate can enforce the development of a rhizosphere specific microbiome for the mutual benefit of plant and microbe. SignificanceThe grain crop sorghum exudes an herbicidal compound called sorgoleone from its root tips, which inhibits the growth of other plants. We isolated bacteria that grow on sorogleone and identified a cluster of bacterial genes required for sorogleone degradation that can be used as a biomarker for this trait. An approach to improve the production of crops in stressful conditions such as drought, is to encourage their association with plant growth promoting bacteria. Our discovery of sorgoleone degradation genes opens the door to engineering bacteria that receive benefit from sorghum in the form of a plant-specific growth substrate, and in return promote the growth of this crop.

microbiology↗

bioRxiv2023

Toll-like receptor 7 (TLR7)-mediated antiviral response protects mice from lethal SARS-CoV-2 infection

SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFN/{beta}) and type III (IFN{lambda}) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.

immunology↗

bioRxiv2023

SpatialData: an open and universal data framework for spatial omics

Spatially resolved omics technologies are transforming our understanding of biological tissues. However, handling uni- and multi-modal spatial omics datasets remains a challenge owing to large volumes of data, heterogeneous data types and the lack of unified spatially-aware data structures. Here, we introduce SpatialData, a framework that establishes a unified and extensible multi-platform file-format, lazy representation of larger-than-memory data, transformations, and alignment to common coordinate systems. SpatialData facilitates spatial annotations and cross-modal aggregation and analysis, the utility of which is illustrated via multiple vignettes, including integrative analysis on a multi-modal Xenium and Visium breast cancer study.

bioinformatics↗

bioRxiv2023

Six3 and Six6 jointly regulate the identities and developmental trajectories of multipotent retinal progenitor cells in the mouse retina

Formation, maintenance, and differentiation of tissue-specific progenitor cells are fundamental tasks during organogenesis. Retinal development is an excellent model for dissecting these processes; mechanisms of retinal differentiation can be harnessed for retinal regeneration toward curing blindness. Using single-cell RNA sequencing of embryonic mouse eye cups in which transcription factor Six3 was conditionally inactivated in peripheral retinas on top of germline deletion of its close paralog Six6 ("DKO"), we identified cell clusters and then inferred developmental trajectories in the integrated dataset. In control retinas, naive retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was directly from naive retinal progenitor cells at G1 phase, and the retinal neuron trajectory was through a neurogenic state marked by Atoh7 expression. Upon Six3 and Six6 dual deficiency, both naive and neurogenic retinal progenitor cells were defective. Ciliary margin differentiation was enhanced, and multi-lineage retinal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Differential expression analysis not only confirmed previous phenotype studies but also identified novel candidate genes regulated by Six3/Six6. Six3 and Six6 were jointly required for balancing the opposing gradients of the Fgf and Wnt signaling in the central-peripheral patterning of the eye cups. Taken together, we identify transcriptomes and developmental trajectories jointly regulated by Six3 and Six6, providing deeper insight into molecular mechanisms underlying early retinal differentiation.

developmental biology↗

bioRxiv2023

Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements

Cis-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time1-3. Here we sought to connect the structural changes at cis-regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate cis-regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.

genomics↗