Biology subjects

Swain, R. K.

Publications and source records attributed to Swain, R. K..

10 recordsLinked to original sources

bioRxiv2026

Rewiring systemic nodulation control to engineer heat-tolerant nitrogen fixation in Medicago truncatula and chickpea

Root nodule symbiosis allows legumes to meet their nitrogen requirements. However, the causes of symbiosis failure at high temperatures remain unknown. We demonstrated that elevated temperatures affect both the host and the microbial partner in Medicago truncatula and chickpea. Rhizobial viability and nod gene expression persisted under heat stress, but plant transcriptome analysis revealed a strong downregulation of the nodule organogenesis machinery. Nodule organogenesis was restored by constitutive overexpression of NIN-LBD16-NFYA1-NFYB16, miR2111, and CLE5 knockdown but failed to restore nitrogen fixation. In contrast, the genetic manipulation of the competence pathway involving CEP peptides significantly improved both nodulation and nitrogen fixation. Constitutive loss of the autoregulation of nodulation (AON) pathway in the SUNN alleles also rescued root nodulation and nitrogen fixation. Therefore, heat stress perturbs the balance between AON and the competence pathway, and genetic manipulation of these pathways is a promising approach to engineer thermotolerant nitrogen fixation in crop legumes.

plant biology↗

bioRxiv2025

FRG1 Regulates Nonsense-Mediated mRNA Decay by Modulating UPF1 Levels

FRG1 acts as a molecular double-edged sword: while its overexpression is linked to facioscapulohumeral muscular dystrophy (FSHD), its under expression activates tumorigenic signalling pathways--highlighting the importance of tightly regulated FRG1 levels. Although prior studies have hinted at FRG1s involvement in RNA biogenesis, its core molecular functions have remained largely elusive. Our previous work identified FRG1 as a transcriptional regulator of genes in the nonsense-mediated decay (NMD) pathway; however, the underlying mechanisms were not fully delineated. In this study, using a range of cellular models with altered FRG1 expression, we demonstrate that reduced FRG1 levels enhance NMD activity. We reveal that FRG1 is a structural component of both the spliceosome and the exon junction complex (EJC), and that it directly modulates NMD by interacting with UPF1--regulating its ubiquitination and degradation. Furthermore, we show that DUX4 inversely regulates the NMD machinery through FRG1, establishing a critical molecular axis. Despite its structural association with the EJC and spliceosome, the absence of FRG1 does not compromise the integrity of these complexes. Polysome profiling showed that FRG1 co-sediments with eIF4A3, a core EJC component, across translating ribosomal fractions. This interaction was further supported by proximity ligation assays confirming the close spatial proximity of FRG1 and eIF4A3. Importantly, we validated the impact of FRG1 perturbation on NMD efficiency and UPF1 levels in vivo using a transgenic FRG1 knockout zebrafish model. Together, these findings establish a direct, DUX4-independent role for FRG1 in regulating the NMD pathway and uncover a previously unrecognized function for FRG1 in post-transcriptional gene regulation. Our work provides mechanistic insight into FRG1s molecular roles and lays the groundwork for therapeutic strategies targeting diseases associated with its dysregulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/689056v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1ef00e1org.highwire.dtl.DTLVardef@22cd63org.highwire.dtl.DTLVardef@da4080org.highwire.dtl.DTLVardef@c0d283_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2025

Cfap300 regulates the transdifferentiation of Corpuscle of Stannius cells in zebrafish

Stanniocalcin 1 (Stc1) is a hormone secreted by the Corpuscle of Stannius (CS) gland in teleost fish, including zebrafish, where it regulates calcium homeostasis. The CS gland forms by transdifferentiation of pronephric tubule epithelial cells at the distal early (DE) and distal late (DL) boundary. Although transdifferentiation is known in development and disease, the underlying mechanisms remain unclear. Here, we show that cfap300, a DNAAF implicated in primary ciliary dyskinesia (PCD), is essential for CS gland formation. TALEN-generated cfap300 mutants develop normally and show no nephron segmentation defects, but exhibit impaired CS development, which could be partially rescued by cfap300 mRNA injection. Further, cfap300 mutants display increased expression of cdh17 driven by Hnf1ba, which blocks CS precursor transdifferentiation. Knockdown of cdh17 or hnf1ba restores CS formation. These results uncover a Cfap300-Hnf1ba-Cdh17 pathway that regulates epithelial-to-endocrine transdifferentiation, revealing a novel, lineage-specific role for a DNAAF protein in endocrine organogenesis.

developmental biology↗

bioRxiv2025

Chikungunya virus susceptibility increases in the absence of mk2b and mk3 genes in zebrafish model

Chikungunya virus (CHIKV) infection imposes a significant socio-economic burden due to the absence of effective antiviral treatments and vaccines. Host factors are essential for the CHIKV life cycle, making them promising targets for antiviral therapy. Previous studies have identified Mitogen-activated protein kinase activated protein kinase 2 (MK2) and Mitogen-activated protein kinase activated protein kinase 3 (MK3) as key host factors in CHIKV infection; however, their role in an animal model remain unclear. This study highlights the critical roles of MK2 and MK3 host factors in an animal model following CHIKV infection. To investigate their functions, mk2b (mk2b-/-), mk3 (mk3-/-), and mk2b-mk3 (mk2b-/-mk3-/-) double knockout zebrafish were generated using the CRISPR-Cas9 technique. A significant high viral titer of CHIKV was observed in case of all knockout groups compared to the wild-type (WT) control using plaque assay, RT-qPCR and immunofluorescence assay. Among the knockout groups, mk3-/- displayed the highest susceptibility to CHIKV, followed by mk2b-/-. In contrast, the mk2b-/-mk3-/- double knockout exhibited the lowest susceptibility to CHIKV infection. Additionally, severe symptoms such as bent body, impaired response to physical stimuli, and increased mortality were most pronounced in mk3-/- larvae compared to other knockouts and the WT. The expression levels of inf{phi}1 and rsad2 were also elevated in all knockout groups during the early days of infection indicating higher interferon response in the absence of mk2b and mk3 during CHIKV infection. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling the CHIKV infection in organism level and subsequently may contribute in designing antiviral therapeutics in future. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections. Author SummaryCHIKV, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, causes febrile illness and has spread across Africa, Asia, Europe, and America. Despite extensive research, effective antiviral drugs and vaccines are yet to be commercially available. This study examines the role of mk2b and mk3 host factors following CHIKV infection. For this purpose, mk2b and mk3 single as well as double knockout have been generated in zebrafish using the CRISPR-Cas-9 technique. Findings suggest that zebrafish exhibit high CHIKV susceptibility in the absence of mk2b and mk3, confirming its importance during infection. Moreover, these three knockout models could serve as a valuable platform for examine the role of mk2b and mk3 in the presence of other viral infection.

molecular biology↗

bioRxiv2024

NEK9 ablation rewires docetaxel resistance through induction of ERK-mediated cancer cell pyroptosis

Docetaxel alone or in combination with other drugs is the most common chemotherapy regimen for several neoplasms including advanced OSCC. Unfortunately, chemoresistance leads to relapse and continued tumor growth. It is therefore important to explore the causative factors for docetaxel resistance. In this study, we performed a CRISPR-based kinome screening that identified Never In Mitosis Gene-A Related Kinase-9 (NEK9) as a major player of docetaxel resistance in OSCC, prostate, and pancreatic cancer lines. NEK9 expression was upregulated in tumor samples of chemotherapy non-responders compared to responder OSCC patients. Our validation data suggests selectively knocking out NEK9 sensitizes cancer cells to docetaxel. Mechanistically, we found that ablation of NEK9 induces DNA damage, activating ERK(p-T202/Y204) that leads to Gasdermin-E mediated Cancer Cell pyroptosis. The in-vitro kinase activity assay identified fostamatinib as a potent inhibitor of NEK9. The xenograft data suggest that fostamatinib restores docetaxel sensitivity and facilitates a significant reduction of tumor burden. Overall, our data suggests a novel combination of fostamatinib and docetaxel needs further clinical investigation in advanced OSCC.

cancer biology↗

bioRxiv2022

CMTM6 mediates cisplatin resistance in OSCC by regulating AKT/c-MYC driven ribosome biogenesis

CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin resistant OSCC lines stably expressing NtShRNA and CMTM6 ShRNA revealed that CMTM6 might be a potential regulator of ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or by pharmacological inhibitor (CX-5461), significantly restores cisplatin medicated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of ribosome biogenesis network and targeting ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.

cancer biology↗

bioRxiv2021

Isolation and Characterization of SARS-CoV-2 strains circulating in Eastern India.

Emergence of SARS-CoV-2 as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts and rapid emergence of new variants urge for establishment of research infrastructure to facilitate rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, five SARS-CoV2 strains (ILS01, ILS02, ILS03, ILS15 and ILS24) of four different clades (19A, 19B, 20A and 20B) were isolated from patient swab samples collected during the 1st COVID-19 wave in Odisha, India. The viral isolates were adapted to in-vitro cultures and further characterized to identify strain specific variations in viral growth characteristics. All the five isolates showed substantial amount of virus induced CPE however ILS03 belonging to 20A clade displayed highest level of CPE. Time kinetics experiment revealed spike protein expression was evident after 16th hours post infection in all five isolates. ILS03 induced around 90% of cytotoxicity. Further, the susceptibility of various cell lines (human hepatoma cell line (Huh-7), CaCo2 cell line, HEK-293T cells, Vero, Vero-E6, BHK-21, THP-1 cell line and RAW 264.7 cells) were assessed. Surprisingly, it was found that the human monocyte cells THP-1 and murine macrophage cell line RAW 264.7 were permissive to all the SARS-CoV-2 isolates. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government run vaccine drive in India. The micro-neutralization assay suggested that both Covaxin and Covishield vaccines were equally effective (100% neutralization) against all of the isolates. The whole genome sequencing of culture adapted viral isolates and viral genome from patient oropharyngeal swab sample suggested that repetitive passaging of SARS-CoV2 virus in Vero-E6 cells did not lead to emergence of many mutations during the adaptation in cell culture. Phylogenetic analyses revealed that the five isolates clustered to respective clades. The major goal was to isolate and adapt SARS-CoV-2 viruses in in-vitro cell culture with minimal modification to facilitate research activities involved in understanding the molecular virology, host-virus interactions, application of these strains for drug discovery and animal challenge models development which eventually will contribute towards the development of effective and reliable therapeutics.

microbiology↗

bioRxiv2021

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host-factors having implication in the disease pathogenesis and severity

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/434371v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1930556org.highwire.dtl.DTLVardef@14376d6org.highwire.dtl.DTLVardef@2f064eorg.highwire.dtl.DTLVardef@1472572_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

miR-142 regulates IL-6 signalling during host immune response to Staphylococcus epidermidis infection in zebrafish

Host immune responses to Staphylococcus epidermidis, a frequent cause of nosocomial infections, are not well understood. We have established a novel bath immersion model of this infection in zebrafish larvae. S.epidermidis infection activates Tlr-2 signalling pathway by upregulation of tlr-2. Macrophages play a primary role in the host immune response and are involved in clearance of infection in the larvae. There is marked inflammation characterised by heightened NF-{kappa}B signalling and elevation of several pro-inflammatory cytokines. Infected larvae show rapid upregulation of il-1b and tnf-a transcripts and relatively slower elevation of il-6 transcription. The IL-6 signalling pathway is additionally subject to amplification by elevation of IL-6 signal transducer (il-6st) levels, which negatively correlates with miRNA dre-miR-142-5p expression. Enhanced IL-6 signalling is protective to the host in this model as inhibition of the signalling pathway resulted in increased mortality upon S.epidermidis infection. Our study describes the host immune responses to S.epidermidis infection, identifies a likely role for miR-142-5p - il-6st interaction in modulating this response and establishes the importance of IL-6 signalling in this infection model.

immunology↗

bioRxiv2019

Allosteric feed-forward activation of AP-2 via FCHO- BMP2K axis promotes clathrin-mediated endocytosis

Spatio-temporal regulation of central adaptor complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). We recently discovered that FCHO proteins trigger clathrin-coated pit (CCP) formation by allosterically activating AP-2 on plasma membrane (Umasankar et al., 2014). Here, we demonstrate that this activation promotes AP-2 phosphorylation via recruitment and stabilization of BMP-2 inducible kinase (BMP2K), a bona fide AP-2 kinase leading to CCP maturation. Accordingly, BMP2K mislocalizes and degrades in FCHO knockout/ AP-2 depleted cells. Functional inactivation of kinase impairs AP-2 phosphorylation leading to altered lattice morphology and CME phenotypes reminiscent of CCP maturation defects. Reexpression of FCHO rescues AP-2 phosphorylation defects in FCHO knockout cells implying membrane activation of AP-2 is a prerequisite for kinase function. Furthermore, gain- and loss-of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions during zebrafish embryogenesis. Together, our findings reveal an in vivo allosteric feed-forward axis for operation of CME.

cell biology↗