Biology subjects

Jain, D.

Publications and source records attributed to Jain, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Unravelling the role of IRX4 variants in non-syndromic and Down syndrome associated congenital heart disease

IRX4 is a TALE- homeodomain transcription factor which is essential for cardiac development. In murine models, Irx4 deficiency leads to impaired ventricular function and results in cardiomyopathy. To elucidate the role of IRX4 in human congenital heart disease (CHD), Sanger sequencing of the IRX4 gene was performed in 205 individuals with non-syndromic CHD, 24 Down syndrome (DS) cases with CHD, 27 DS cases without CHD, and 150 healthy control individuals. Two novel (p.Ser24Asn and p.Thr217Iso) and one reported variant (rs2232376) were identified in non-syndromic CHD. Concurrently, rs2232376 was also detected in DS with CHD. The first novel (p.Ser24Asn) and reported (rs2232376) variants lie in the N-terminal region while the second novel (Thr217Iso) variant lies within the TALE homeodomain. In silico structural modelling suggested that both the novel variants (p.Ser24Asn and Thr217Iso) induce conformational changes in the IRX4 protein, potentially altering its DNA-binding affinity. A significant reduced expression of IRX4 muteins was noted in Western blotting by both variants (p.Ser24Asn and Thr217Iso). Furthermore, luciferase reporter assays demonstrated decline in the activity of Nanog promoter and HEY2 enhancer in response to both the variants which was further corroborated by decrease mRNA expression in qRT-PCR. Additional downstream targets, including Nfyc, Nppa, and Bmp10, also exhibited anomalous expression due to both the variants (p.Ser24Asn and Thr217Iso). Altogether, the aberrant expression of muteins as well as downstream target genes along with compromised activities of promoters substantiate the pathogenic potential of the identified IRX4 variants and underscore the critical role of IRX4 in regulating multiple stages of cardiogenesis.

genetics↗

bioRxiv2026

An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

RALPH (RNase-like proteins associated with haustoria) effectors, which are preferentially expressed in haustoria and structurally resemble fungal T1/F1 RNases, constitute one of the largest effector families in powdery mildew (PM) fungi, yet their functions in dicot-adapted PM species remain poorly understood. Unlike cereal PM RALPHs, which lack the catalytic residues required for RNase activity, some dicot PM RALPHs retain these residues. Here, we performed a comprehensive structural and expression-based characterization of the pea PM Erysiphe pisi RALPH (EpRALPH) repertoire and functionally characterized EpRALPH11, a RALPH effector with partial conservation of the catalytic residues of T1/F1 fungal RNases. Comparative analyses identified multi-RNase-domain RALPHs as a conserved feature of the Erysiphe lineage, while expression profiling showed that many EpRALPHs are preferentially expressed in haustoria during early host colonization. AlphaFold 3-based structural analyses revealed a conserved T1/F1 RNase-like fold despite substantial sequence and surface charge divergence, indicating functional diversification among EpRALPHs. EpRALPH11 enhanced susceptibility to E. pisi in Medicago truncatula, localized to the nucleolus, and induced nucleolar fragmentation when heterologously expressed in Nicotiana benthamiana leaves. Its RNase domain exhibited T1 RNase activity in vitro, supporting the retention of a catalytically competent RNase domain and, together with its nucleolar localization, suggesting that EpRALPH11 targets plant rRNA and disrupts nucleolar functions. The RNase domain induced cell death in N. benthamiana, whereas the full-length protein and catalytic mutants did not. Cell death induction required exclusive nucleolar localization of the RNase domain, and an extended N-terminal intrinsically disordered region suppressed this activity in the full-length protein. Together, our findings reveal a previously unrecognized mechanis regulating RNase activity in a dicot PM RALPH effector and provide new insights into the functional diversification of RALPHs and their adaptation to obligate biotrophy.

plant biology↗

bioRxiv2026

Dual-Method Immune Deconvolution Reveals Subtype-Specific PD-L1 Drivers and a lower case Greek gammaT Cell M2 Macrophage Axis in Breast Cancer

Most computational studies of the breast cancer immune microenvironment rely on a single deconvolution algorithm. Since different tools analyze different parameters, integrated information across a dataset can be missed. Here, we employed two complementary approaches, xCell and a CIBERSORT-style ssGSEA using LM22 signatures, on 1,099 PAM50-classified primary tumors from the TCGA-BRCA cohort. PD-L1 was highest in basal-like tumors (Kruskal-Wallis p < 0.001) and correlated with CD8+ T cells ({rho} = 0.65) and M1 macrophages ({rho} = 0.67) in that subtype, which fits the standard model of IFN-{gamma}-driven adaptive upregulation. In HER2-enriched cancers, PD-L1 tracked with both effector and regulatory populations simultaneously, while luminal tumors were largely immune-quiet. The most consequential finding involved {gamma}{delta} T cells and M2 macrophages: xCell showed a non-significant correlation ({rho} = 0.048, p = 0.11), whereas CIBERSORT-ssGSEA, using curated {gamma}{delta} gene signatures, produced a significant correlation ({rho}= 0.565, p < 2.2 x 10-{superscript 1}) that held across all five subtypes. A multivariate model explained 49% of PD-L1 variance (adjusted R{superscript 2} = 0.49), and Cox regression incorporating immune features gave a concordance of 0.60. These results suggest a baseline {gamma}{delta}-M2 immunosuppressive circuit in breast cancer, particularly in ER+ disease, that could be useful to set a therapeutic target.

cancer biology↗

bioRxiv2026

Deciphering the role of the non-active site ancillary residues in maintaining the activity and substrate specificity of OXA-232 beta-lactamase

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

molecular biology↗

bioRxiv2026

Critical roles of MCM8 in meiotic recombination during mouse spermatogenesis

Meiotic DNA double-strand break (DSB) formation and repair by homologous recombination is crucial for ensuring proper chromosome segregation. In mice, the mini-chromosome maintenance family protein, MCM8, has been proposed to function in meiotic recombination and its loss leads to infertility, but the underlying mechanisms are poorly understood. Here we used cytological and genomic assays to infer the role of MCM8 during meiotic recombination in mouse spermatocytes. We show that MCM8-deficient spermatocytes exhibit increased levels of SPO11-dependent DSBs at recombination hotspots during early prophase. DSBs are resected normally and accumulate strand-exchange proteins. However, downstream recombination intermediates are barely detected and recombination intermediate-associated MutSgamma foci do not form efficiently. Consistent with a role in early recombination intermediate processing, MCM8 binds to displacement loop (D-loop) structures in vitro. We propose that MCM8 controls meiotic recombination in at least two ways. MCM8 participates in regulating meiotic DSB number. Further, MCM8 plays a role in the formation and/or stability of post-resection recombination intermediates, steps that are critical for DSB repair via recombination and for efficient synapsis of homologous chromosomes during mouse meiosis.

molecular biology↗

bioRxiv2026

Coronin1A regulates tumor microenvironment in colitis-associated colorectal cancer in a SUMO-dependent way

Inflammatory bowel disease (IBD), comprising Crohns disease and Ulcerative colitis, is a group of multifactorial illnesses with persistent gastrointestinal inflammation and a series of undesirable consequences. IBD patients have a three times higher risk of developing colitis-associated colorectal cancer (CAC). A higher mutational burden due to persistent inflammation acts as a driver of dysplasia and even tumorigenesis. While the pathological sequence is known, the molecular mechanisms underlying the transition from chronic inflammation to CAC remain largely elusive. Post-translational modification- SUMOylation plays an integral role in shaping gut inflammation as well as several forms of cancers, including sporadic colorectal cancer. In this study, the contribution of SUMOylation to CAC pathogenesis was characterized. In the AOM-DSS CAC mice model and human IBD patient specimens, SENP5, but not other deSUMOylases, shows an altered expression. Notably, both SENP5 expression dynamics and alterations in the SUMOylome occur in chronically inflamed and neoplastic colon tissues. SENP5 interactome analysis identified Coronin1A (Coro1A), an actin-binding protein predominantly expressed in immune cells. Coro1A also shows a state-specific, distinct expression pattern in the colon. Interestingly, in contrast to wild-type mice, Coro 1A knockout mice were resistant to polyp formation, with reduced cell proliferation, oncogene expression, and epithelial-to-mesenchymal transition (EMT) gene activation, and reduced extracellular matrix (ECM) development and fibrosis, suggesting its integral role in tumorigenesis. WT mice with chronically inflamed colons and polyps have a higher number of M2-like macrophages, with increased abundance of Coro1A, suggesting a role of Coro1A in modulating the tissue microenvironment toward a pro-tumorigenic state. Mechanistically, Coro1A physically interacts with TGF-{beta} RI and regulates TGF-{beta}-TGF-{beta} RI signalling endosome stability, thereby controlling TGF-{beta}-mediated macrophage polarization. Detailed in vitro experiments revealed stabilization of Coro 1A through its interaction with SUMOylated Raftlin protein. Overall, Coro 1A is necessary and sufficient for TGF-{beta} signalling, macrophage polarization, and tumorigenesis in CAC.

cancer biology↗

bioRxiv2025

Ewing Sarcoma Breakpoint Region 1 (EWSR1) protein binds to the non-structural protein-2 and the non-coding regions of the Chikungunya virus genome to inhibit the virus replication

Chikungunya virus (CHIKV) is an alphavirus with a single-stranded RNA genome. Conserved sequence elements within the 5'- and 3'-non-coding regions (NCRs) of the CHIKV genome interact with host proteins to regulate viral RNA replication. Electrophoretic mobility shift assays (EMSA) and mass spectrometry identified several host RNA-binding proteins that associate with these conserved NCR elements, among which EWSR1 uniquely bound the 3'-ends of both the plus-sense genomic RNA and the minus-sense replication intermediate RNA. EMSA and biolayer interferometry demonstrated that EWSR1 binds strongly to two RNA elements: a 48-nucleotide region in the plus-strand 3'-NCR (3NCR48) and a 50-nucleotide region at the 3'-end of the minus-strand replication intermediate (5NCR-RC50), which is complementary to the genomes 5'-NCR and its adjacent sequence. RNA immunoprecipitation from CHIKV-infected cells confirmed that EWSR1 binds CHIKV RNA in vivo. Functional analyses revealed that CHIKV infection downregulates EWSR1 expression and causes its cytoplasmic accumulation. Knockdown or knockout of EWSR1 expression increased CHIKV genomic RNA levels and viral titers, whereas its overexpression suppressed both. EWSR1 reduced the synthesis of both plus- and minus-sense CHIKV RNAs in infected cells. Additionally, EWSR1 was found to bind CHIKV nsP2 and inhibit its helicase activity, which is critical for viral genome replication. The antiviral activity of EWSR1 extended to Ross River virus but not to Sindbis virus, consistent with conservation of the 48-nucleotide sequence in their respective 3'-NCRs. Together, these findings identify EWSR1 as a novel host restriction factor that limits CHIKV replication through specific interactions with viral RNA and the nsP2 protein. IMPORTANCEChikungunya virus (CHIKV) poses a significant medical challenge, necessitating the development of novel antiviral strategies. This study demonstrates that the host protein EWSR1 plays a key role in controlling CHIKV replication. By binding to specific regions of viral RNA and interacting with nsP2, a critical viral enzyme, EWSR1 inhibits viral RNA synthesis and reduces viral titers. In the absence of EWSR1, CHIKV replicates more efficiently, underscoring its antiviral function. These findings establish EWSR1 as a novel host restriction factor that limits CHIKV replication. Understanding this interaction enhances our knowledge of host-virus dynamics and may guide the design of new antiviral approaches against CHIKV and related alphaviruses.

microbiology↗

bioRxiv2025

Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

Maintenance of naive pluripotency requires core transcription factors (TFs) like SOX2 and auxiliary TFs like NANOG, yet molecular mechanisms governing their intra-nuclear dynamics and DNA binding interactions remain unclear. Here, using high-density 3D single-molecule light-field microscopy combined with novel spatiotemporal analysis pipelines, we track SOX2 and NANOG dynamics in live cells. Despite lower protein abundance, NANOG displays a similar chromatin-bound fraction to SOX2. This arises partially because, while both TFs undergo frequent transient non-specific binding interactions ([~]0.5-0.7s), NANOG exhibits more stable specific binding ([~]25s vs [~]16s). Both TFs also assemble into phase-separated domains of [~]400 nm containing both freely diffusing and chromatin-bound proteins, which further influences their dynamics. Strikingly, NANOGs protein-protein interaction domain markedly increases chromatin residence time (>5-fold) and the size of these phase-separated domains. Our work uncovers how NANOG and SOX2 stabilise gene regulatory networks that maintain naive pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics.

cell biology↗

bioRxiv2025

Identification and functional characterization of LDB3 gene variants in DCM patients from Indian Population

The LIM domain-binding protein 3 (LDB3), also known as Z-band alternatively spliced protein (ZASP), is a key component of the Z-disc, essential for maintaining sarcomeric integrity and transmitting contractile force in cardiomyocytes. Genetic variants in LDB3 have been implicated in cardiomyopathies, including dilated cardiomyopathy (DCM). In this study, we have screened 100 idiopathic DCM cases along with controls by Sanger sequencing and identified nine non-synonymous (p.S184I, p.D193N, p.K204R, p.R229C, p.P295T, p.Q402P, p.F465I, p.F496L, p.P606S), seven synonymous (p.T91=, p.A152=, p.D168=, p.S182=, p.A279=, p.S347=, p.A358=), and seven intronic (c.322-50G>C, c.548+53A>C, c.718+47G>C, c.718+81G>A, c.755+11G>A, c.755+86G>A, c.*30C>G) variants, among which six (p.S184I, p.D193N, p.P295T, p.F465I, p.F496L, p.P606S) missense variants were novel. In-vitro studies demonstrated that p.K204R increased LDB3 expression whereas p.S184I, p.F465I, p.F496L, p.P606S reduced LDB3 expression. Immunostainning revealed cytoplasmic aggregation of the p.K204R-LDB3 protein. The variant p.R229C also over-expressed the mutant protein. In case of variant, p.P295T, Z-disc was severely disrupted indicating impaired Z-disc integrity. Furthermore, synonymous variants showed altered mRNA folding, stability, and codon usage bias, potentially affecting translation efficiency. In-silico analyses predicted p.F465I, p.F496L, p.P606S, and p.S184I variants to be deleterious, significantly altering protein structure. Structural modeling using AlphaFold2 showed high RMSD values, suggesting conformational destabilization. Collectively, these findings highlight LDB3 as a hypermutable candidate gene for DCM in our cohort that may contribute to DCM pathogenesis by perturbing protein structure, expression, and cellular localization, underscoring the critical role of LDB3 in cardiac muscle function and disease.

genetics↗

bioRxiv2025

Enhancing CRISPR Education: A Plug-And-Play Framework for Teaching Gene Editing in Plant Biology

Modern molecular biology tools and technologies such as CRISPR have sped up scientific discovery. From an educational perspective, these advancements are both exciting and overwhelming. Educators shaping these future scientists face the ongoing challenge of staying compliant with the latest developments in molecular biology while finding effective ways to teach these discoveries. As the use of CRISPR gene editing technology continues to expand globally, there is an increasing need for a workforce that is both knowledgeable about its theoretical foundations and trained in its practical use. While advanced, technology-driven STEM courses have the potential to improve student retention, they are often lecture-heavy and lack intentional engagement strategies that support deeper learning. Moreover, agriculture is the second most impacted sector by this technology, yet there is a significant lack of teaching materials focused on CRISPR in plant biology. To address these gaps, we developed a framework for teaching gene editing that incorporates multiple engagement strategies beyond traditional lecture-based instruction. This framework was implemented over two semesters in an Introduction to Gene Editing course at Tennessee State University, offered to both undergraduate and graduate students enrolled in a degree in Agricultural Sciences. This manuscript outlines the various strategies used in the course including active learning, multimodal instructional approaches and experiential learning strategies that can be adopted in other classrooms to effectively teach gene editing. Survey-based results from the course indicate a measurable increase in student comfort with designing and executing CRISPR-Cas based experiments. Societal Impact StatementPlant biology lacks accessible teaching materials for CRISPR, a powerful gene-editing technology widely used to improve agriculture. We developed an engaging framework to teach CRISPR concepts to advanced undergraduates and graduate students in plant sciences, which can be readily adopted by other instructors. The approach increased self-reported confidence in students and comfort in explaining CRISPR. Since instructors often have limited time to design interactive lessons, this framework offers a ready-to-use, effective strategy that makes CRISPR more widely available in classrooms, ultimately strengthening CRISPR literacy in the future agricultural workforce.

scientific communication and education↗

bioRxiv2025

Emetine dihydrochloride inhibits Chikungunya virus nsP2 helicase and shows antiviral activity in the cell culture and mouse model of virus infection

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes frequent epidemics of chikungunya fever across the world and has become a significant medical challenge, warranting the development of novel antivirals. A cell imaging-based high-content screening of the Spectrum collection of small molecules, containing all the US and international drug molecules, identified Emetine dihydrochloride (ED) as a potent antiviral against CHIKV. ED inhibited CHIKV replication in the C57BL/6 mouse model of chikungunya disease, resulting in significantly lower viremia. Importantly, clinical symptoms of joint swelling were altogether absent in the CHIKV-infected mice treated with ED. In the ERMS cells, ED inhibited the CHIKV uptake, and the virus replication early during the virus life cycle by inhibiting the viral RNA synthesis, resulting in the inhibition of the viral protein synthesis. ED was predicted to bind the helicase domain of the CHIKV non-structural protein nsP2 by in silico methods. The ED binding to the nsP2 protein and its helicase domain was validated in vitro by microscale thermophoresis and isothermal titration calorimetry. Importantly, ED inhibited the CHIKV nsP2 helicase activity in vitro in a dose-dependent manner. These data demonstrate the potential of ED to be repurposed as a novel CHIKV antiviral, warranting clinical development. AUTHOR SUMMARYChikungunya virus (CHIKV) spreads through the bites of the virus-infected Aedes mosquitoes. The virus causes frequent epidemics of chikungunya fever involving severe joint edema and muscle pain. The virus activity has been recorded in more than 100 countries in Asia, Africa, South and Central America, and Europe, and it has become endemic in several regions of the world. In 2024, around 480,000 CHIKV cases and over 200 deaths were reported worldwide. While a CHIKV vaccine was recently approved, no virus-specific antiviral therapy is available. Considering the global medical significance of the virus, the development of effective CHIKV antivirals is a priority. With a view to repurposing an existing drug, we screened a collection of all the approved US and international drug molecules and identified Emetine dihydrochloride (ED) as a potent inhibitor of CHIKV replication in the cell culture model. The CHIKV-infected mice treated with ED showed reduced virus replication and no joint swelling, which is a typical clinical symptom of CHIKV disease. Our studies show that ED binds the CHIKV nsP2, inhibiting its helicase action, which is required for replicating the viral genomic RNA and producing the viral proteins. In the past, emetine has been used in humans to treat amoebiasis. Our work thus shows the potential of the drug emetine to be repurposed for treating CHIKV patients.

microbiology↗

bioRxiv2024

Conserved ancillary residues situated proximally to the VIM-2 active site affect its metallo β-lactamase activity

Verona-integron-metallo-{beta}-lactamase (VIM-2) is one of the most widespread class B {beta}-lactamase, responsible for {beta}-lactam resistance. Although active-site residues help in metal binding, the residues nearing the active-site possess functional importance. Here, to decipher the role of such residues in the activity and stability of VIM-2, the residues E146, D182, N210, S207, and D213 were selected through in-silico analyses and substituted with alanine using site-directed mutagenesis. The effects of substitution mutations were assessed by comparing the changes in the {beta}-lactam susceptibility pattern of E. coli host cells expressing VIM-2 and its mutated proteins. VIM-2_N210A enhanced the susceptibility of the host by [~]4-8 folds against penicillins and cephalosporins while the expression of VIM-2_D182A radically increased the susceptibility of the host. However, expression of VIM-2_E146A reduced the susceptibility of the host by 2-fold. Further, proteins were purified to homogeneity, and VIM_N210A and VIM_D182A displayed reduced thermal stability than VIM-2. Moreover, in vitro catalytic efficiencies of VIM-2_D182A were drastically reduced against all the {beta}-lactams tested whereas the same were moderately reduced for VIM-2_N210A. Conversely, the catalytic efficiency was marginally altered for VIM_E146A. Overall, we infer that both N210A and D182A substitutions negatively affect the performance of VIM-2 by influencing substrate specificity and stability, respectively.

microbiology↗

bioRxiv2024

Acinetobacter baumannii DacC influences cell shape, biofilm formation, and physiological fitness by manifesting DD-carboxypeptidase and β-lactamase dual-enzyme activities

With the growing threat of drug-resistant Acinetobacter baumannii, there is an urgent need to comprehensively understand the physiology of this nosocomial pathogen. As penicillin-binding proteins are attractive targets for antibacterial therapy, herein we have tried to explore the physiological roles of two putative DD-carboxypeptidases, viz., dacC and dacD in A. baumannii. Surprisingly, the deletion of dacC resulted in a reduced growth rate, loss of rod-shaped morphology, reduction in biofilm-forming ability, and enhanced susceptibility towards {beta}-lactams, whereas, the deletion of dacD had no such effect. Interestingly, ectopic expression of dacC restored the lost phenotypes. The double deletion mutant in which both dacC and dacD were absent showed properties similar to the dacC single knockout. On the other hand, cell-shape reverting efficiency in septuple PBP deleted E. coli and in vitro enzyme kinetics assessments reveal that dacD is a stronger DD-CPase as compared to dacC. The expression of dacC was in the log phase whereas dacD expression takes place in the stationary phase. In summary, we conclude that dacC encodes a dual enzyme, possessing activities of DD-CPase and {beta}-lactamase, which significantly affects the physiology of A. baumannii in various ways whereas dacD encodes a strong DD-CPase and plays a role in cell morphology, though it exerts negligible impact on other physiological aspects like intrinsic antibiotic resistance or biofilm formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/603720v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18820c9org.highwire.dtl.DTLVardef@208baeorg.highwire.dtl.DTLVardef@b2673eorg.highwire.dtl.DTLVardef@983aae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

A Multitrait Genome-Wide Association Study Reveals a Requirement for the Strigolactone Receptor DWARF14 in Optimal GOLVEN10 Signaling

GOLVEN/ROOT MERISTEM GROWTH FACTOR family of signaling peptides have been shown to control root lateral organ number, density and positioning in plants, although the signaling pathways involved remain obscure. A diverse set of 171 Medicago truncatula HapMap accessions with variation in responses to the GOLVEN 10 peptide, GLV10, were used to identify 74 significant loci controlling seven traits related to nodule formation and root architecture. Importantly, a single nucleotide polymorphism (SNP) in the upstream region of the MtGLV10 peptide-inducible strigolactone receptor gene, MtDWARF14 was significantly associated with insensitivity of nodule density to GLV10, suggesting a link between strigolactone signaling and GLV10 responsiveness. Three independent d14 mutants of the DWARF14 gene were found to hypernodulate, while overexpression of the gene led to reduction in nodule number, phenocopying GLV10. A null mutant, mtd14-1, remained sensitive to GLV10s effect on nodule density. However, at the transcriptional level, the mutant failed to effectively induce the expression of the GOLVEN marker genes, MtPLETHORA3 and MtPINLIKES2. Our study uncovers a hitherto unknown link between the strigolactone and GLV peptide signaling pathways using genotype x environment analysis of Medicago HapMap lines and provides a putative molecular mechanism for recovery from frost damage to fine roots.

plant biology↗

bioRxiv2024

Microarray Integrated Spatial Transcriptomics (MIST) for Affordable, Robust, and Comprehensive Digital Pathology

10X Visium, a popular Spatial transcriptomics (ST) method, faces limited adoption due to its high cost and restricted sample usage per slide. To address these issues, we propose Microarray Integrated Spatial Transcriptomics (MIST), combining conventional tissue microarray (TMA) with Visium, using laser-cutting and 3D printing to enhance slide throughput. Our design facilitates independent replication and customization in individual labs to suit specific experimental needs. We provide a step-by-step guide from designing TMAs to the library preparation step. We demonstrate MISTs cost-effectiveness and technical benefits over Visium and GeoMx Nanostring. We also introduce AnnotateMap, a novel computational tool for efficient analysis of multiple ROIs processed through MIST.

bioinformatics↗

bioRxiv2024

Two retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity

The human genome contains 24 gag-like capsid genes derived from deactivated retrotransposons conserved among eutherians. Although some of their encoded proteins retain the ability to form capsids and even transfer cargo, their fitness benefit has remained elusive. Here we show that the gag-like genes PNMA1 and PNMA4 support reproductive capacity. Six-week-old mice lacking either Pnma1 or Pnma4 are indistinguishable from wild-type littermates, but by six months the mutant mice become prematurely subfertile, with precipitous drops in sex hormone levels, gonadal atrophy, and abdominal obesity; overall they produce markedly fewer offspring than controls. Analysis of donated human ovaries shows that expression of both genes declines normally with aging, while several PNMA1 and PNMA4 variants identified in genome-wide association studies are causally associated with low testosterone, altered puberty onset, or obesity. These findings expand our understanding of factors that maintain human reproductive health and lend insight into the domestication of retrotransposon-derived genes.

genetics↗

bioRxiv2024

Anatomy of a pest control failure: Introgression of cytochrome P450 337B3 alleles from invasive old-world bollworm into native corn earworm.

The establishment of invasive species populations can threaten the ecological balance in naive habitats and impact agricultural production practices. Helicoverpa armigera (old-world bollworm, OWBW) and H. zea (corn earworm, CEW) were geographically separated prior to the 2013 report of OWBW invasion into South America. Introgression of OWBW-specific cytochrome P450 337B3 (CYP337B3) gene into CEW was repeatedly detected across South America and the Caribbean. Two hybrids were documented from Texas in 2019. In this study, screening insects collected in Olathe, Colorado, USA, where a failure of pyrethroids to control CEW damage to conventional sweetcorn in 2023 detected 28.6% of insects with the OWBW-specific CYP337B3 marker. Nucleotide sequencing of the CYP337B3 gene identified 73.1 and 26.9% of insects carried CYP337B3v2 and CYP337B3v6 alleles, respectively and 0.15 overall frequency of CYP337B3 alleles. Based on prior data for distinct phylogeographic origins of CYP337B3v2 and v6 alleles, our results indicate Olathe samples were derived from two different introductions; An uncertain source of the v6 allele that was initially reported in West Africa and possibly South American or Caribbean origin of the globally distributed v2 allele. One of the 1618 individuals screened also carried a ribosomal RNA internal transcribed spacer 1 (ITS1) derived from OWBW. Local selection pressures at the Olathe location imposed by repeated pyrethroid exposures are likely attributed to the prevalence of CYP337B3, where control practices hasten the accumulation of phenotypic resistance by adaptive introgression. Pyrethroid and other resistance factors carried by invasive OWBW may continue to impact CEW management tactics across the Americas.

genetics↗

bioRxiv2023

Evaluation of morpho-physiological and yield-associated traits of rice (Oryza sativa L.) landraces combined with marker-assisted selection under high temperature stress and elevated atmospheric CO2 levels

Rice (Oryza sativa L.) has a tremendous domestication history and is presently used as a major cereal all over the world. In Asia, India is considered as one of the centers of origin of indica rice and has several native landraces, especially in North-Eastern India (NEI), which have the potential to cope with the negative impact of present-day climate change. The current investigation aimed to evaluate the NEI rice landraces potential under high temperatures and elevated CO2 levels in comparison with a check variety for phenological, morphological, physiological and yield-associated parameters and molecularly validated with marker-assisted genotyping. The initial experiment was carried out with 75 rice landraces to evaluate their high heat tolerance ability. Seven better-performing landraces along with the check variety (N22) were further evaluated for aforesaid traits across two years (2019 and 2020) under control (or T1) and two stress treatments - (i) mild stress or T2 [CO2 550 ppm + 4{degrees} C more than ambient temperature] and (ii) severe stress or T3 [CO2 750 ppm + 6{degrees} C more than ambient temperature] using bioreactors. In the molecular analysis, the eight selected genotypes were evaluated through 25 Start Codon Targeted (SCoT) markers. The results revealed that the mild stress (T2) had a positive impact on various morpho-physiological parameters like plant height, number of leaves, leaf area and yield parameters like spikelets panicle-1 (S/P), thousand-grain weight (TGW) and grain yield (GY). This effect could be attributed to the genotypes ability to maintain a higher photosynthetic rate and possess better tolerance ability to moderately high temperatures. However, under high-temperature conditions in T3, all genotypes exhibited a significant decrease in the studied parameters including GY. It was found that pollen traits were significantly and positively correlated to spikelet fertility% at maturity, which was further significantly associated with GY under applied stress conditions. The physiological traits including shoot biomass were evident to have a significant positive effect on yield-associated parameters like S/P, harvest index (HI), TGW and GY. Overall, two landraces Kohima special and Lisem were found to be better responsive compared to other landraces as well as the check variety N22 under stress conditions. SCoT genotyping amplified a total of 77 alleles out of which 55 were polymorphic with the PIC value ranging from 0.22 to 0.67. The investigation suggests the presence of genetic variation among the tested rice lines and further supports evidence of the closely relatedness of Kohima special and Lisem. These two are two better-performing rice landraces from North-East India based on their improving morpho-physiological parameters and yield attributes in mild and severe high temperature and elevated CO2 stress environments. The shortlisted two rice landraces can be used as valuable pre-breeding materials for future rice breeding programs to improve the stress tolerance properties, particularly to high temperatures and elevated CO2 levels under ongoing changing climatic scenarios.

plant biology↗