Biology subjects

Poddar, S.

Publications and source records attributed to Poddar, S..

10 recordsLinked to original sources

bioRxiv2026

Amaranthus viridis-derived phytopriming reprograms redox homeostasis and limits arsenic accumulation in rice

Arsenic (As) poses a major threat to rice productivity and food safety due to its high bioaccumulation potential and subsequent entry into the human food chain. In rice, As impairs seed germination, disrupts morpho-anatomical development, and induces oxidative stress. This study evaluates seed priming with an aqueous extract of the agricultural weed Amaranthus viridis (AvE) as a sustainable strategy to alleviate As-induced phytotoxicity. AvE priming significantly improved germination (71-75%) and morpho-physiological performance under As stress. It reduced oxidative stress markers, including H2O2 (21-38%), malondialdehyde (13-26%), and proline (18.9-44.7%), while increasing antioxidant metabolites, polyphenols and glutathione by up to 2.34-fold and 41%, respectively. Microscopy confirmed restoration of cellular integrity and anatomical organisation in primed seedlings. ICP-OES analysis showed that AvE priming reduced root As uptake by up to 39%, root-to-shoot translocation by up to 58%, and grain As accumulation by up to 95% compared with unprimed plants. qRT-PCR revealed modulation of genes involved in As homeostasis, indicating coordinated physiological and transcriptional responses. Importantly, improved agronomic performance further demonstrated the translational potential of this approach. This study provides the first evidence that A. viridis extract is a cost-effective, sustainable biostimulant for producing low-As rice in contaminated regions.

plant biology↗

bioRxiv2026

Assessing the physiological S nitrosoproteome reveals nitric oxide mediated regulatory networks in rice

Nitric oxide metabolism-based protein post-translational modifications, such as reversible S- nitrosylation, have been at the pinnacle of plant redox research owing to their significant correlation with seed dormancy, interaction with other signaling molecules, plant development and metabolism, biotic and abiotic plant stress responses, immune defense responses against plant pathogens, and senescence. The rapid interconversion of reactive nitrogen species, the abrogation of nitric oxide homeostasis by exogenous supplementation of NO donors and scavengers, the lack of spatio-temporal specificity of NO signaling, and the limited bioavailability or assay sensitivity for detection often limit the effectiveness of identifying and characterizing S-nitrosothiols in plants. Hitherto unknown, we report the first experimental evidence of the total in vivo S-nitrosoproteome in Oryza sativa L. subsp. indica, comprising 134 PSNOs, enriched with 169 putative sites susceptible to S-nitrosylation, without any exogenous supplementation of NO donors. In the present study, mercuric salt- driven facile decomposition of S-nitrosoproteins in the presence of nitrone spin trap 5,5- dimethyl-1-pyrroline N-oxide, resulting in the synthesis of DMPO-nitrone adducts with PSNO-derived protein thiyl radicals in O. sativa, has been demonstrated as an efficient and novel strategy for characterizing the PSNOs using mass spectrometry analysis. The evidence of physiological levels of PSNOs was further re-examined in a bi-directional qualitative and quantitative approach involving the 2,3-diaminonaphthalene assay in tandem with fluorescence-based visualization and fluorometric quantification. In silico analyses, involving both functional enrichment and pathway prediction analyses, have furthermore revealed unique protein-protein interaction networks and signaling pathways among the S- nitrosoproteome candidates and their predictable physiological roles in O. sativa indica, awaiting further in vitro validation for their functional correlation in response to S- nitrosylation. In conclusion, the present study provides novel evidence of nitric oxide signaling in rice cultivars under physiological conditions, bringing new insights into the potential in vivo transnitrosylation of regulatory or active-site cysteine thiols.

biochemistry↗

bioRxiv2026

A role for heavy chain-modification in protecting hyaluronan from free radical fragmentation during inflammation

The glycosaminoglycan hyaluronan (HA) is an essential and ubiquitous component of human tissues and biofluids. The only known covalent modification of HA entails the attachment of heavy chains (HC) from the inter-alpha-inhibitor (II) family of proteoglycans, forming stable complexes (HC*HA) that arise during inflammation. In some contexts, HC*HA is thought to contribute to pathology, whereas in others it may form part of a protective pathway. However, its exact roles are not fully understood. Here, we report that HC modifications can protect HA from fragmentation by reactive oxygen species (ROS) produced during the inflammatory cascade. Using solid-state nanopore molecular size analysis, we show that HA is highly resistant to degradation from exogenous ROS in vitro when in the context of HC*HA complexes, while the unmodified HA polymer is fragmented rapidly under the same conditions. Experiments performed with admixtures of HA and unbound antioxidant proteins - including HC-bearing components - demonstrate the necessity of covalent HC attachment to the polysaccharide for the protection. Finally, we find that HA with high-HC content from inflammatory equine synovial fluid has increased resilience to ROS damage compared to low-HC HA from a healthy joint. Collectively, these results demonstrate that covalent HC modification is an effective biological strategy for preserving HA integrity against ROS fragmentation, including in inflammatory conditions.

molecular biology↗

bioRxiv2025

Role of small intronic RNAs in the crosstalk between immune cells and beta-cells during type 1 diabetes development

Small non-coding RNAs, such as microRNAs and tRNA-derived fragments, are key regulators of cellular processes, but the functions of small intronic RNAs (sinRNAs), a recently identified RNA class, remain largely unknown. Here, we report that two sinRNAs, sinR-D and sinR-T, are upregulated in pancreatic {beta}-cells of NOD mice, a well-established model of type 1 diabetes. Using in vivo RNA-tagging, we demonstrate that these sinRNAs are packaged into extracellular vesicles released by infiltrating CD4 T lymphocytes and subsequently delivered to {beta}-cells during the early stages of autoimmune attack. Functional analyses revealed that overexpression of sinR-T has little effect on {beta}-cell viability, whereas sinR-D markedly increases {beta}-cell apoptosis. This finding suggests that the transfer of sinR-D contributes to {beta}-cell destruction and the onset of type 1 diabetes. Furthermore, pull-down experiments with biotinylated sinRNAs identified Ago2, a core component of the RNA-induced silencing complex (RISC), as a binding partner of sinR-D, indicating mechanistic parallels with microRNA-mediated regulation. Collectively, our data uncover a novel role for sinRNAs as extracellularly transferred regulators of {beta}-cell fate, expanding the repertoire of small RNAs implicated in the initiation of type 1 diabetes.

molecular biology↗

bioRxiv2022

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes the highly mutagenic uracil lesion from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence, yet the underlying principles that dictate UNG substrate specificity have remained elusive. Here, we show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates containing AUT, TUA, AUA, and TUA motifs. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. A strong correlation between UNG efficiency and substrate flexibility was observed, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. The finding that substrate flexibility controls UNG efficiency has implications in diverse fields, including the genesis of mutation hotspots, molecular evolution, and understanding sequence preferences of emerging base editors.

biophysics↗

bioRxiv2022

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation rate bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 m gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34{degrees}C for 24 hours resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34{degrees}C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

plant biology↗

bioRxiv2022

Impact of temperature and time on DNA-free Cas9-ribonucleoprotein mediated gene editing in wheat protoplasts and immature embryos

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes that result in insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

plant biology↗

bioRxiv2020

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

plant biology↗

bioRxiv2019

Defective Nucleotide Catabolism Defines a Subset of Cancers Sensitive to Purine Nucleoside Phosphorylase Inhibition

Small molecule inhibitors of purine nucleoside phosphorylase (PNP) have been explored as a treatment strategy for leukemia and lymphoma, however, the determinants of response to this class of drugs are incompletely understood. PNP inhibitors impair cell proliferation by preventing catabolism of the nucleoside deoxyguanosine (dG) which induces toxic imbalances amongst intracellular deoxyribonucleotide triphosphate (dNTP) pools following its phosphorylation and trapping by nucleoside kinases. We hypothesized that differential nucleoside uptake or catabolism defines cancer cell lines as either sensitive or resistant to PNP inhibition. Among cancer cell lines we found that T-cell acute lymphoblastic leukemia (T-ALL) cells are uniquely and acutely sensitive to PNP inhibition whereas the B-cell leukemia and solid tumor models are completely resistant. We determined that although the nucleoside scavenging kinase deoxycytidine kinase (dCK) was active in all cells tested, PNP inhibitors only induced dGTP pool increases in sensitive models. By evaluating the expression of key genes involved in nucleotide scavenging, biosynthesis, and phosphohydrolysis in a panel of sensitive and resistant cell lines we found that the dNTP phosphohydrolase SAM histidine aspartate containing protein 1 (SAMHD1) was exclusively expressed in resistant models. Using CRISPR/Cas9 SAMHD1 knockout cell lines, we verified that PNP inhibitor sensitivity is a function of SAMHD1 expression and determined that the pharmacological inhibition of dCK or genetic restoration of SAMHD1 conferred resistance to PNP inhibition. Importantly, we determined that low expression of SAMHD1 is not limited to T-ALL as subset of established and primary solid tumors models are SAMHD1-deficient. These solid tumor models were consistently acutely sensitive to PNP inhibitors which indicates that the utility of PNP inhibitors extends beyond hematological malignancies. Additionally, we found that deoxycytidine (dC) can limit the anti-proliferative effects of PNP inhibitors by competing with dG for phosphorylation by dCK but this effect can be overcome by expression of dC-catabolizing gene cytidine deaminase (CDA). Collectively, these results indicate that SAMHD1, dCK and CDA are critical biomarkers that must be used to stratify patients in clinical trials evaluating pharmacological PNP inhibition.

cancer biology↗

bioRxiv2018

Kluyveromyces marxianus as a robust synthetic biology platform host

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits- thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

synthetic biology↗