Biology subjects

Thapa, R.

Publications and source records attributed to Thapa, R..

14 recordsLinked to original sources

bioRxiv2026

Capturing Spatial Heterogeneity Using UAV-Derived Multispectral Phenotypes and Random Regression Models Improves Genetic Evaluation in Alfalfa Breeding Trials

Spatial heterogeneity is a major source of environmental noise in field trials, reducing genetic evaluation accuracy. Conventional spatial correction methods use field layout and plot proximity but often fail to capture in-season dynamics or leverage temporal data from high-throughput phenotyping. We hypothesized that longitudinal UAV-derived multispectral measurements could characterize field-scale environmental heterogeneity and improve genetic evaluation in breeding trials. Multispectral imagery was collected throughout the growing season across eight alfalfa trials in New York and New Mexico (20222023). Vegetation indices were evaluated for heritability and association with biomass yield using univariate and multi-trait models. Longitudinal measurements were modeled with random regression (RR) to estimate plot-specific permanent environmental effects (PEE), incorporated into genomic mixed models within a two-stage framework. Model performance was compared with conventional autoregressive (AR) and baseline (no correction) models using metrics of consistency and accuracy of estimated genetic effects across replicates. Vegetation indices showed moderate to high heritability, with GNDVI consistently outperforming NDVI and NDRE. Both univariate and multi-trait analyses revealed substantial spatial variability, particularly in New Mexico trials. RR-derived PEE captured field-scale spatial patterns and improved genetic signal recovery relative to baseline and AR models, with the largest gains under strong spatial heterogeneity and comparable performance where variability was limited. The RR framework improved reproducibility of genetic effect estimates across independent field subsets without compromising agreement with full-trial evaluations. These findings show that integrating UAV data via random regression enhances genetic signal recovery and selection accuracy, with broader potential for precision agriculture and biosensor-driven crop management.

genetics↗

bioRxiv2026

Prevention of Unc13a cryptic splicing is sufficient to preserve memory

TDP-43 dysfunction is thought to underlie frontotemporal dementia and limbic-predominant age-related TDP-43 encephalopathy, neurodegenerative dementias currently without effective therapy. Therapeutic strategies are designed to correct individual cryptic targets of TDP-43, such as UNC13A, whereby its cryptic splicing compromises synaptic function, yet the sufficiency of such an approach to prevent memory deficits is unclear. Using a forebrain neuron-specific TDP-43 knockout mouse model that recapitulates TDP-43 dysfunction occurring during early stages of human disorders, we found here that prevention of cryptic splicing to include that of Unc13a attenuated memory deficits. We show that genetic ablation of Unc13a cryptic exon solely in such TDP-43 knockout mice is sufficient to preserve cognition, supporting the clinical value of targeting UNC13A to mitigate memory deficits. Prevention of cryptic splicing of multiple targets of TDP-43 additionally attenuate neuron loss. For optimal outcomes in TDP-43 related dementias, these findings thus strongly support strategies designed to repress cryptic splicing of multiple targets of TDP-43, including UNC13A.

pathology↗

bioRxiv2026

(2R,6R)-Hydroxynorketamine elicits rapid antidepressant effects by promoting astrocytic μ-δ opioid receptor heterodimerization

Ketamine produces rapid antidepressant effects but is constrained by psychotomimetic properties and abuse potential. The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) shows antidepressant-like efficacy without N-methyl-D-aspartate receptor (NMDAR) blockade, yet its upstream targets remain unclear. Here we show that HNK potentiates hippocampal excitatory transmission and reverses stress-induced behavioural deficits through opioid receptor signaling. Pharmacological and genetic analyses reveal a requirement for both {micro}- and {delta}-opioid receptors in astrocytes. Chronic stress reduces {micro}-{delta} receptor heterodimers in the hippocampus, and a single dose of HNK restores their abundance. PAINT-MINFLUX nanoscopy quantifies increased {micro}-{delta} heterodimerization, and molecular dynamics simulations indicate selective binding of HNK to the {micro}-receptor protomer via Asp147 and Tyr148. Mutating these residues abolishes HNK-driven heterodimer formation, downstream signaling and rapid antidepressant-like effects in vivo. Astrocytic {micro}-{delta} opioid receptor heterodimers thus represent a targetable mechanism for next-generation rapid-acting antidepressants.

neuroscience↗

bioRxiv2025

Identification and characterization of the delta-12 fatty acid desaturase from Euglena gracilis

Fatty acid desaturase 12 (FAD12) is a key enzyme in fatty acid biosynthesis, responsible for converting oleic acid to linoleic acid through desaturase activity. Euglena gracilis (Euglena) is an emerging platform for the industrial production of various metabolites, including lipids. However, a comprehensive understanding of Euglenas fatty acid biosynthesis pathways remains incomplete, posing a significant barrier to the commercialization of Euglena bioproducts. To address this gap, we employed a bioinformatics approach to identify a Euglena gracilis FAD12 (Eg FAD12). We analyzed the evolutionary relationship of Eg FAD12 with its homologs from other organisms and revealed that the three canonical histidine box motifs are conserved among FAD12s. To characterize EgFAD12, we cloned it into the pEAQ-hyperstrans vector and overexpressed it in Nicotiana benthamiana to take advantage of its endogenous fatty acid pool, which could act as substrates. The heterologous expression of FAD12 in N. benthamiana led to an increased linoleic acid content, demonstrating the suspected desaturase activity. To further confirm the function of Eg FAD12, we performed CRISPR-Cas9-mediated knockout of Eg FAD12 in Euglena, which resulted in a drastic reduction in linoleic acid (C18:2) without compromising biomass yield or lipid content. This work advances our understanding of fatty acid biosynthesis in Euglena and will aid in its adoption as a platform for producing customized lipids.

microbiology↗

bioRxiv2025

Broad brain biodistribution conferred by an AAV to restore TDP-43 function mitigates Frontotemporal Demenia-like deficits

TDP-43 dysfunction is an early pathogenic determinant of frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), a devastating disorder currently without effective therapy. Here, we exploit a blood-brain-barrier (BBB)-permeable AAV (AAV-PHP.eB) that confers broad brain biodistribution to restore TDP-43 function in a TDP-43 deficient model (CamKIIa-CreER;Tardbp mice) that mimics the early stage of TDP-43 dysfunction occurring in FTLD-TDP. Intracerebroventricular delivery by AAV-PHP.eB of CTR, our previously characterized splicing repressor, revealed its accumulation in [~]40% of adult hippocampal neurons. Remarkably, treatment of adult CamKIIa-CreER;Tardbpf/fmice with AAV-PHP.eB-CTR restored TDP-43 function, attenuated neuronal aberrant activity and memory deficits, and rescued neuron loss. Importantly, we showed that TDP-43s autoregulatory element restricts CTR expression to a physiological range. No overt phenotype was observed after long-term exposure to AAV-PHP.eB-CTR in aged mice, highlighting a favorable safety profile for this gene therapy. These results validate that BBB-crossing AAVs can deliver CTR with a biodistribution in the adult brain that is broad enough to rescue FTD-like phenotypes, supporting clinical testing of this gene therapy for FTLD-TDP.

neuroscience↗

bioRxiv2025

Extracellular Vesicles from Multiple Sclerosis White Matter Exhibit Synaptic, Mitochondrial, Complement, and Aging-related Pathway Dysregulation

Extracellular vesicles (EVs) are increasingly recognized as mediators of central nervous system (CNS) function and pathologies, including multiple sclerosis (MS). While plasma-derived EVs have been explored as biomarkers in MS, little is known about EVs in CNS tissue. Here, we characterize EVs from postmortem white matter (WM) of MS and control brains. EVs were separated by differential centrifugation followed by size exclusion chromatography and characterized using nanoflow cytometry, single-particle reflectance imaging sensing (SP-IRIS), and transmission electron microscopy. EV size, yield, and morphology did not differ significantly between MS and control samples. Proteomic analyses revealed downregulation of synaptic and mitochondrial proteins and upregulation of complement and inflammatory proteins and pathways in MS WM EVs. This suggests that EVs reflect ongoing synaptic pathology, metabolic dysfunction, and CNS-compartmentalized inflammation and that they may actively contribute to these pathological processes. Deconvolution analyses suggests a shift in EV cellular origin, with an increased astrocytic and decreased neuronal EV contributions in MS. Several proteomic changes we observed in CNS-derived EVs have also been reported in circulating EVs of people with MS, establishing this CNS tissue EV study as a valuable resource for identifying biomarker candidates for brain-derived plasma EV studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/657509v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f88da5org.highwire.dtl.DTLVardef@11b7ee9org.highwire.dtl.DTLVardef@291d9forg.highwire.dtl.DTLVardef@157cb73_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

The fungal peptide toxin candidalysin induces distinct membrane repair mechanisms compared to bacterial pore-forming toxins

The common fungal pathogen, Candida albicans, relies on the pore-forming toxin candidalysin to damage host cells. Cells counteract pore-forming toxins by Ca2+-dependent mechanisms, such as microvesicle shedding and annexin recruitment to resist cholesterol-dependent cytolysins like streptolysin O (SLO), or annexin involvement and patch repair in the case of aerolysin. However, the specific Ca2+-dependent repair pathways engaged in response to candidalysin remain poorly understood. Here, we determined the involvement of different Ca2+-dependent repair mechanisms to candidalysin and compared responses to SLO and aerolysin using flow cytometry and high-resolution microscopy. We report that candidalysin triggered Ca2+-dependent repair, but patch repair and ceramide failed to provide significant protection. MEK-dependent repair and annexins A1, A2 and A6 contributed partially to repairing damage caused by candidalysin. However, annexin translocation after candidalysin challenge was delayed compared to SLO or aerolysin challenge. Surprisingly, extracellular Cl- improved cell survival after candidalysin challenge, but not after challenge with SLO or aerolysin. Finally, we found that candidalysin is removed via extracellular vesicle shedding. These findings reveal that Ca2+-dependent microvesicle shedding protects cells from candidalysin and can be engaged by multiple molecular mechanisms during membrane repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653080v2_figa1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@433b3aorg.highwire.dtl.DTLVardef@1e583f3org.highwire.dtl.DTLVardef@139f086org.highwire.dtl.DTLVardef@de07b0_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Candidalysin is resisted by distinct repair mechanisms compared to bacterial PFTs.After pore formation and membrane damage by each toxin, multiple repair pathways are triggered downstream of Ca{superscript 2} flux. Candidalysin induces a protective Cl- influx and activates MEK-dependent repair, which contributes to cell protection. Annexin translocation occurs slowly and provides minor protection, while patch repair is ineffective. In contrast, aerolysin does not benefit from Cl- influx or MEK protection. Aerolysin triggers moderate annexin translocation and relies primarily on patch repair as the main protective mechanism. Streptolysin O elicits rapid annexin translocation and activates MEK signaling, both of which contribute to robust protection. Patch repair plays only a minor protective role against SLO. The figure was created using BioRender. C_FIG

cell biology↗

bioRxiv2024

Remote sensing for estimating genetic parameters of biomass accumulation and modeling stability of growth curves in alfalfa

Multi-spectral imaging (MSI) collection by unoccupied aerial vehicles (UAV) is an important tool to measure growth of forage crops. Information from estimated growth curves can be used to infer harvest biomass and to gain insights in the relationship of growth dynamics and harvest biomass stability across cuttings and years. In this study, we used MSI to evaluate Alfalfa (Medicago sativa L. subsp. sativa) to understand the longitudinal relationship between vegetative indices (VIs) and forage/biomass, as well as evaluation of irrigation treatments and genotype by environment interactions (GEI) of different alfalfa cultivars. Alfalfa is a widely cultivated perennial forage crop grown for high yield, nutritious forage quality for feed rations, tolerance to abiotic stress, and nitrogen fixation properties in crop rotations. The direct relationship between biomass and VIs such as Normalized difference vegetation index (NDVI), green normalized difference vegetation index (GNDVI), red edge normalized difference vegetation index (NDRE), and Near infrared (NIR) provide a non-destructive and high throughput approach to measure biomass accumulation over subsequent alfalfa harvests. In this study, we aimed to estimate the genetic parameters of alfalfa VIs and utilize longitudinal modeling of VIs over growing seasons to identify potential relationships between stability in growth parameters and cultivar stability for alfalfa biomass yield across cuttings and years. We found VIs of GNDVI, NDRE, NDVI, NIR and simple ratios to be moderately heritable with median values for the field trial in Ithaca, NY to be 0.64, 0.56, 0.45, 0.45 and 0.40 respectively, Normal Irrigation (NI) trial in Leyendecker, NM to be 0.3967, 0.3813, 0.3751, 0.3239 and 0.3019 respectively, and Summer Irrigation Termination (SIT) trial in Leyendecker, NM to be of 0.11225, 0.1389, 0.1375, 0.2539 and 0.1343, respectively. Genetic correlations between NDVI and harvest biomass ranged from 0.52 - .99 in 2020 and 0.08 - .99 in 2021 in the NY trial. Genetic correlations for NI trial in NM for NDVI ranged from 0.72 - .98 in 2021 and SIT ranged from 0.34-1.0 in 2021. Genotype by genotype by interaction (GGE) biplots were used to differentiate between stable and unstable cultivars for locations NY and NM, and Random regression modeling approaches were used to estimate growth parameters for each cutting. Results showed high correspondence between stability in growth parameters and stability, or persistency, in harvest biomass across cuttings and years. In NM, the SIT trial showed more variation in growth curves due to stress conditions. The temporal growth curves derived from NDVI, NIR and Simple ratio were found to be the best phenotypic indices on studying the stability of growth parameters across different harvests. The strong correlation between VIs and biomass present opportunities for more efficient screening of cultivars, and the correlation between estimated growth parameters and harvest biomass suggest longitudinal modeling of VIs can provide insights into temporal factors influencing cultivar stability.

genetics↗

bioRxiv2023

Optimal investments in private land conservation depend more on landholder preferences than climate change

Effective private land conservation strategies that consider both landholder preferences and future climatic conditions are critical for preserving biodiversity and ecosystem services. Yet, the interaction and relative importance of these factors for conservation planning performance is unknown. Here, we assess the importance of considering landholder preferences and climate change for prioritising locations for conservation tenders to recruit landholders for conservation covenants. To achieve this we develop a planning framework that accounts for the tender process to optimise investment across regions and apply it to koala-focused tenders in New South Wales (NSW), Australia, exploring four planning approaches that consider or are ignorant to landholder preferences and the tender process and/or climate change. We find that optimal investments depend more on landholder preferences than climate change, and when landholder preferences are ignored, there is little benefit in accounting for climate change. Our analysis reveals new insights into this important interaction.

ecology↗

bioRxiv2023

Pathogenic Mutations in the C2A Domain of Dysferlin form Amyloid that Activates the Inflammasome.

Limb-Girdle Muscular Dystrophy Type-2B/2R is caused by mutations in the dysferlin gene (DYSF). This disease has two known pathogenic missense mutations that occur within dysferlins C2A domain, namely C2AW52R and C2AV67D. Yet, the etiological rationale to explain the disease linkage for these two mutations is still unclear. In this study, we have presented evidence from biophysical, computational, and immunological experiments which suggest that these missense mutations interfere with dysferlins ability to repair cells. The failure of C2AW52R and C2AV67D to initiate membrane repair arises from their propensity to form stable amyloid. The misfolding of the C2A domain caused by either mutation exposes {beta}-strands, which are predicted to nucleate classical amyloid structures. When dysferlin C2A amyloid is formed, it triggers the NLRP3 inflammasome, leading to the secretion of inflammatory cytokines, including IL-1{beta}. The present study suggests that the muscle dysfunction and inflammation evident in Limb-Girdle Muscular Dystrophy types-2B/2R, specifically in cases involving C2AW52R and C2AV67D, as well as other C2 domain mutations with considerable hydrophobic core involvement, may be attributed to this mechanism.

biophysics↗

bioRxiv2022

Patch repair protects cells from the small pore-forming toxin aerolysin

Small pore-forming toxins in the aerolysin family lyse cells by damaging the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four membrane repair mechanisms have been proposed: toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which of these repair mechanisms aerolysin triggers is unknown. Furthermore, Ca2+ flux triggered by aerolysin is controversial, yet membrane repair responses require Ca2+. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Since aerolysin triggered Ca2+ flux, we investigated Ca2+-dependent repair pathways. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to CDCs, expression of the patch repair protein dysferlin potently protected cells from aerolysin. We propose that aerolysin triggers a Ca2+-dependent death mechanism that obscures repair responses, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms.

cell biology↗

bioRxiv2022

Rapid Activation of Hematopoietic Stem Cells

Adult Hematopoietic Stem Cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation to accelerate the production of needed effector blood and immune cells. Surprisingly, little is known about the earliest stages of HSC activation. We utilize surface markers of HSC activation, CD69, and CD317, revealing response as early as 2 h after stimulation. The dynamic expression of HSC activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify the dose response, demonstrating a low-threshold and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence into proliferation. Our data show that the premier response of adult stem cells to immune stimulation is rapid, sensitive, and directly leads to proliferation.

immunology↗

bioRxiv2019

Meiosis and Kinetochore genes are used by cancer cells as genome destabilizers and transformation catalysts

Cancer cells have an altered transcriptome which contributes to their altered behaviors compared to normal cells. Indeed, many tumors express high levels of genes participating in meiosis or kinetochore biology, but the role of this high expression has not been fully elucidated. In this study we explore the relationship between this overexpression and genome instability and transformation capabilities of cancer cells. For this, we obtained expression data from 5 different cancer types which were analyzed using computational information-theoretic analysis. We were able to show that highly expressed meiotic/kinetochore genes were enriched in the altered gene expression subnetworks characterizing unstable cancer types with high chromosome instability (CIN). However, altered subnetworks found in the cancers with low CIN did not include meiotic and kinetochore genes. Representative gene candidates, found by the analysis to be correlated with a CIN phenotype, were further explored by transfecting genomically-stable (HCT116) and unstable (MCF7) cancer cell lines with vectors overexpressing those genes. This overexpression resulted in an increase in the numbers of abnormal cell divisions and defective spindle formations and in increased transformation properties in stable cancer HCT116 cells. Interestingly, the same properties were less affected by the overexpressed genes in the unstable MCF7 cancer cells. Our results indicate that overexpression of both meiosis and kinetochore genes is capable of driving genomic instability and cancer progression.

cancer biology↗

bioRxiv2018

NetrinG1/NGL-1 Axis promotes pancreatictumorigenesis through cancer associated fibroblastderived nutritional supply and immunosuppression

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SignificancePDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

cancer biology↗