Biology subjects

Pradhan, M.

Publications and source records attributed to Pradhan, M..

10 recordsLinked to original sources

bioRxiv2024

QTL mapping in field plant populations reveals a genetic basis for frequency- and spatially-specific fungal pathosystem resistance

Fungal pathogens pose significant challenges to agro-ecosystem productivity. The wild tobacco, Nicotiana attenuata, has been grown for over two decades at an experimental field station in its native habitat, leading to the emergence of a high-mortality sudden wilt disease caused by a Fusarium-Alternaria pathosystem. By using an Advanced Intercross Recombinant Inbred Line (AI-RIL) mapping population of N. attenuata planted in the infected field site, we found two significant loci associated with plant susceptibility to the fungi. A functional characterization of several genes in these loci identified RLXL (intracellular ribonuclease LX-like) as an important factor underlying plant pathogen resistance. Virus-induced silencing of RLXL reduced leaf wilting in plants inoculated with an in vitro culture of Fusarium species. Assessing the significance of the RLXL-associated allele in mixed field populations indicated that, among 4-plant subpopulations, those harboring a single plant with the RLXL-deficiency allele exhibited the highest survival rates. Within these populations, a living RLXL-deficient plant improved the survival of RLXL-producing plants located diagonally, while the mortality of the adjacent plants remained as high as in all other subpopulations. Taken together, these findings provide evidence for the genetic basis for a frequency- and spatially-dependent population pathogen resistance mechanism. Significance statementPlant pathogen resistance studies predominantly focus on single genes that reduce pathogenicity in individual plants, aiming to apply these findings to agricultural monocultures. On the other hand, ecologists have observed for decades that greater diversity drives plant population resistance and resilience to pathogens. More studies are needed to identify and characterize loci with positive effects conferred through their frequency in plant populations. We combine quantitative genetics, molecular techniques, and ecologically-informed mixed field populations to identify a novel intracellular ribonuclease LX-like (RLXL) gene with a frequency- and position-dependent effect for plant resistance. To our knowledge, this is the first detailed link between plant population protection and various percentages of plants with an allele representing RLXL presence or absence.

plant biology↗

bioRxiv2023

Super-resolution histology of paraffin-embedded samples via photonic chip-based microscopy

Fluorescence-based super-resolution optical microscopy (SRM) techniques allow the visualization of biological structures beyond the diffraction limit of conventional microscopes. Despite its successful adoption in cell biology, the integration of SRM into the field of histology has been deferred due to several obstacles. These include limited imaging throughput, high cost, and the need for complex sample preparation. Additionally, the refractive index heterogeneity and high labeling density of commonly available formalin-fixed paraffin-embedded (FFPE) tissue samples pose major challenges to applying existing super-resolution microscopy methods. Here, we demonstrate that photonic chip-based microscopy alleviates several of these challenges and opens avenues for super-resolution imaging of FFPE tissue sections. By illuminating samples through a high refractive-index waveguide material, the photonic chip-based platform enables ultra-thin optical sectioning via evanescent field excitation, which reduces signal scattering and enhances both the signal-to-noise ratio and the contrast. Furthermore, the photonic chip provides decoupled illumination and collection light paths, allowing for total internal reflection fluorescence (TIRF) imaging over large and scalable fields of view. By exploiting the spatiotemporal signal emission via MUSICAL, a fluorescence fluctuation-based super-resolution microscopy (FF-SRM) algorithm, we demonstrate the versatility of this novel microscopy method in achieving superior contrast super-resolution images of diverse FFPE tissue sections derived from human colon, prostate, and placenta. The photonic chip is compatible with routine histological workflows and allows multimodal analysis such as correlative light-electron microscopy (CLEM), offering a promising tool for the adoption of super-resolution imaging of FFPE sections in both research and clinical settings.

pathology↗

bioRxiv2022

Gut bacteria impact host uric acid burden and its association with atherosclerosis

Humans with metabolic and inflammatory diseases, including atherosclerosis harbor dysbiotic gut communities. However, the microbes and microbial pathways that influence disease progression remain largely undefined. Here, we show that variation in atherosclerosis burden is in part driven by the gut microbiota and it is associated with circulating levels of the proinflammatory molecule uric acid both in mice and humans. We identify bacterial taxa present in the gut spanning multiple phyla, including Bacillota (Firmicutes), Fusobacteriota and Pseudomonadota (Proteobacteria), that use uric acid and adenine- a key precursor of nucleic acids in intestinal cells, as carbon and energy sources anaerobically, and uncover a gene cluster encoding key steps of purine degradation that is widely distributed among gut dwelling bacteria. Furthermore, we demonstrate that colonization of germ-free mice with purine-degrading bacteria modulates levels of uric acid and other purines in the gut and systemically. Altogether this work demonstrates that gut microbes are important drivers of host global purine homeostasis and uric acid levels, and suggests that gut bacterial catabolism of purines may represent a novel mechanism by which the gut microbiome influences host health.

microbiology↗

bioRxiv2021

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

synthetic biology↗

bioRxiv2021

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

pharmacology and toxicology↗

bioRxiv2020

Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present miCLIP2 in combination with machine learning to significantly improve m6A detection. The optimised miCLIP2 results in high-complexity libraries from less input material. Importantly, we established a robust computational pipeline to tackle the inherent issue of false positives in antibody-based m6A detection. The analyses are calibrated with Mettl3 knockout cells to learn the characteristics of m6A deposition, including m6A sites outside of DRACH motifs. To make our results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP2 data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification. HighlightsO_LImiCLIP2 produces complex libraries to map m6A RNA modifications C_LIO_LIMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sites C_LIO_LIMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KO C_LIO_LIm6A modifications occur outside of DRACH motifs and associate with alternative splicing C_LI

genomics↗

bioRxiv2020

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

neuroscience↗

bioRxiv2020

Targeting heparan sulfate proteoglycan-assisted endocytosis as a COVID-19 therapeutic option

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-CoV and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, facilitates the attachment of viral particles to the cell surface to promote cell entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry, and reveals drugs capable of targeting this important step in the viral life cycle.

cell biology↗

bioRxiv2020

An OpenData portal to share COVID-19 drug repurposing data in real time

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform - named the NCATS OpenData Portal (ODP) - for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.

microbiology↗

bioRxiv2020

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.One Sentence Summary Blocking SARS-CoV-2 cytopathic effects with selective autophagy inhibitors underlying the clinical benefits of chloroquine and hydroxychloroquine.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗