Biology subjects

Dwivedi, V.

Publications and source records attributed to Dwivedi, V..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering plants tolerant to the toxic proline mimic azetidine-2-carboxylic acid through co-option of a bacterial detoxification mechanism

Azetidine-2-carboxylic acid (Aze) is a toxic, non-proteogenic amino acid produced in restricted plants. It can enter the human food chain through direct or indirect dietary exposure and has been linked to neurological disorders due to its ability to misincorporate for its structural analog proline (Pro) during translation and lead to protein misfolding. Although several microorganisms have evolved mechanisms to detoxify Aze, these pathways have not previously been exploited to engineer Aze tolerance in plants. Here, we heterologously expressed a bacterial Aze hydrolase (HAS) in Arabidopsis thaliana to establish a metabolic strategy for Aze detoxification in plants. Transgenic plants exhibited markedly enhanced tolerance to Aze, as demonstrated by improved root growth, cotyledon development, and survival compared with wild-type plants under Aze stress. We further showed that HAS localized to the cytosol, where it converted Aze into the non-toxic metabolite 2-hydroxy-4-aminobutyrate (HAB). HAB formation was confirmed both in planta and in vitro, demonstrating that HAS remained catalytically active in plant cells and effectively detoxified Aze. Enzyme kinetics revealed that HAS has a high affinity for Aze and is stereospecific for L-Aze, with no activity toward D-Aze. Multi-omics analysis including transcriptomics and misincorporation proteomics show that the transgenic lines misincorporate Aze significantly less compared to wild-type leading to enhanced Aze tolerance. Our findings establish bacterial HAS as an effective metabolic detoxification system that confers enhanced Aze tolerance in plants. This study provides a promising strategy for engineering plant resistance to toxic non-proteogenic amino acids.

plant biology↗

bioRxiv2026

The proteotoxicity of azetidine 2-carboxylic acid is associated with reactive oxygen species accumulation

Plants make diverse metabolites to outcompete neighboring organisms for space and resources. Some of these toxic metabolites broadly disrupt conserved molecular mechanisms, such as protein biosynthesis. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites that interfere with protein biosynthesis. The proline (Pro) analog azetidine-2-carboxylic acid (Aze) inhibits plant growth through misincorporation during protein biosynthesis. However, it is unknown if a cascade of downstream stress responses is triggered following Aze misincorporation. Here, we investigate the morphological and stress responses in Arabidopsis grown on Aze. Investigation of root morphological responses show not only reduced root growth, but increased root branching following growth on Aze. Altered root morphology is coupled with a reduced gravitropic response. Aboveground organs were also affected by Aze, including reduced chlorophyll content, reduced photosynthetic efficiency, and increased anthocyanin content. We then tested whether Aze induces reactive oxygen species (ROS) accumulation using multiple approaches and observed both immediate and sustained accumulation of general ROS and H2O2 following treatment with Aze. When plants were grown on Aze supplemented with Pro, ROS levels were restored to normal levels, suggesting that reducing misincorporation events results in less downstream stress responses. In summary, we find that following Aze treatment a cascade of downstream stress responses is induced that exacerbates the effects of toxic NPAAs. This study sheds light on the mechanism of action of NPAAs and provides information on the downstream consequences of translational errors.

plant biology↗

bioRxiv2026

Population-Scale Landscape of Mobile Element Insertions Across 82 Diverse Indian Populations

Mobile element insertions (MEIs) are a major source of structural variation in human DNA that can affect gene regulation and disrupt coding sequences. Population-scale MEI catalogs remain disproportionately skewed toward European-ancestry cohorts, with limited representation from India despite comprising nearly one-sixth of the global population and encompassing thousands of genetically distinct, largely endogamous communities. We characterized non-reference Alu and LINE1 insertions in 7,478 whole genomes from the GenomeIndia project, representing 82 populations selected to capture Indias geographic, linguistic, and social diversity. We cataloged 26,977 Alu and 7,075 LINE1 insertions, identifying 18,982 previously unreported insertions absent from existing publicly available MEI databases. Insertions bore signatures of purifying selection, depleted in exons and active chromatin and enriched in quiescent regions. MEI variants recapitulated the population structure seen in SNP data. Regulatory variation associated with MEIs was strongly population-structured, with a substantial fraction of European-defined MEI-eQTLs absent in the GenomeIndia cohort or carried at sharply different frequencies across communities, and MEIs in high linkage disequilibrium with GWAS variants. Sixty-nine insertions fell within exons, including a truncating LINE1 in CYP2J2 carried across five populations and a community-specific Alu in CABYR with a founder-effect signature. Collectively, these results reveal a layer of population-specific mobile element variation in Indian populations that is not captured by existing European-anchored reference resources. These variants represent potentially novel sources of genetic variation contributing to lack of transferability of disease associations across populations.

genomics↗

bioRxiv2025

Mon1-Rab7 axis is essential for transport, localization and anchoring of oskar mRNA

Asymmetric localization of oskar mRNA to the posterior of the oocyte is a complex process driven by autonomous and non-cell autonomous mechanisms. The former includes Oskar protein that reinforces localization and anchoring of its mRNA through activation of endocytosis and regulation of actin cytoskeleton; the latter includes signals from the posterior follicle cells (PFCs) that regulates microtubule orientation for polarized transport. Here we identify Monensin Sensitivity 1 (Mon1), as a novel factor regulating anterior-posterior (A-P) patterning. Mon1 is an evolutionarily conserved activator of Rab7-a key regulator of the endo-lysosomal pathway. Embryos lacking maternal mon1 (mon1m) show mislocalized oskar and bicoid mRNAs leading to loss of patterning and lethality. In the mutant oocyte Staufen appears clumpy and the levels of Oskar protein and Par-1 is significantly reduced. Abnormal actin rings are seen in the ooplasm. Driving expression of mon1 in the germline rescues these phenotypes and restores viability. In contrast, expression in the PFC predominantly rescues the Par-1 phenotype with a modest effect on viability. We demonstrate that oskar mRNA interacts with Rab7 suggesting possible role for the Mon1-Rab7 axis in the transport of oskar. We show that Mon1 in the PFCs, regulates PIP2 levels to influence accumulation of Par-1 in the oocyte. We propose that Mon1 regulates oskar localization in two distinct ways: cell autonomously in the germline by regulating Rab7, and non-cell autonomously through the PFCs by regulating accumulation of Par-1. Summary statementMon1, an established Rab converter, has roles in embryonic axial patterning, modulating transport, localisation and anchoring of posteriorly localised mRNA during oocyte maturation. Mon1 influence is both cell autonomous, from within the oocyte and non-cell autonomous, through posterior follicle cells.

developmental biology↗

bioRxiv2025

Single dose alum adjuvanted RBD protein vaccine provides protection against homologous challenge with SARS-CoV-2 Washington strain and heterologous rechallenge with Delta and Omicron BA.5 variants in K18 hACE2 mouse model

Most COVID-19 vaccination strategies require at least two doses-a primary vaccine followed by a booster dose with the updated variant-specific vaccine. However, vaccine and booster dose hesitancy make it challenging to administer multiple doses of vaccines in the unvaccinated and vaccinated populations, respectively. Thus, it is important to determine if vaccinated individuals exposed to SARS-CoV-2 infection develop immune responses that may protect them against emerging variants of concern (VoCs). We have developed mouse models to understand the protective efficacy of an RBD-based vaccine against challenge and rechallenge with SARS-CoV-2 VoCs. Mice were vaccinated with RBD protein vaccine formulated in 2% Alhydrogel (alum) adjuvant by subcutaneous route. To determine the efficacy of RBD vaccine, mice were challenged approximately 4 weeks post-vaccination with SARS-CoV-2 variants by intranasal route. To determine the efficacy of RBD vaccine against SARS-CoV-2 rechallenge, mice were rechallenged with SARS-CoV-2 Delta and Omicron BA.5 variants at day 14 post SARS-CoV-2 Washington (WA) strain challenge. Our data suggest that single dose alum adjuvanted RBD protein from Wuhan strain provides protection against homologous challenge with SARS-CoV-2 WA strain but failed to provide protection against heterologous challenge with Delta and Omicron BA.5 variants. Interestingly, vaccinated mice that survived homologous challenge with the WA strain showed protection against heterologous rechallenge with Delta and omicron BA.5 variants. Furthermore, infectious viral loads of Delta and Omicron BA.5 were not detected in the lung tissues collected from the rechallenged mice at 3 days post-rechallenge. The data suggest that a single dose RBD vaccine from the ancestral Wuhan strain together with survival from WA strain challenge induces protective immune responses against Delta and Omicron BA.5 variants rechallenge. These mouse models will be useful to determine the immune responses that correlate with protection against challenge and rechallenge with SARS-CoV-2 VoCs.

microbiology↗

bioRxiv2025

A Metabolic Complex Involved in Tomato Specialized Metabolism

Specialized metabolites mediate diverse plant-environment interactions. Although, recent work has begun to enzymatically characterize entire plant specialized metabolic pathways, little is known about how different pathway components organize and interact within the cell. Here we use acylsugars - a class of specialized metabolites found across the Solanaceae family - as a model to explore cellular localization and metabolic complex formation of pathway enzymes. These compounds consist of a sugar core decorated with acyl groups, which are connected through ester linkages. In Solanum lycopersicum (tomato) four acylsugar acyltransferases (SlASAT1-4) sequentially add acyl chains to specific hydroxyl positions on a sucrose core leading to accumulation of tri and tetraacylated sucroses in the trichomes. To elucidate the spatial organization and interactions of tomato ASATs, we expressed SlASAT1-4 proteins fused with YFP in N. benthamiana and Arabidopsis protoplasts. Our findings revealed a distributed ASAT pathway with SlASAT1 and SlASAT3 localized to the mitochondria, SlASAT2 localized to the cytoplasm and nucleus, and SlASAT4 localized to the endoplasmic reticulum. To explore potential pairwise protein-protein interactions in acylsugar biosynthesis, we used various techniques, including co-immunoprecipitation, split luciferase assays, and bimolecular fluorescence complementation. These complementary approaches based on different interaction principles all demonstrated interactions among the different SlASAT pairs. Following transient expression of SlASAT1-4 in N. benthamiana, we were able to pull down a complex consisting of SlASAT1-4, which was confirmed through proteomics. Size exclusion chromatography of the SlASAT pulldown suggests a heteromultimeric complex consisting of SlASATs and perhaps other proteins involved in this interaction network. This study sheds light on the metabolic coordination for acylsugar biosynthesis through formation of an interaction network of four sequential steps coordinating efficient production of plant chemical defenses.

biochemistry↗

bioRxiv2024

Human melanoma cell lines that possess wild-type BRAF alleles but are dependent on ERBB4 and ERBB2

Metastatic skin cutaneous melanomas that contain wild-type BRAF alleles typically possess an activating mutation in a RAS allele or a loss-of-function mutation in an NF1 allele ("BRAF-WT&RAS/NF1-mutant melanomas"). Nonetheless, these tumors remain a significant clinical challenge; they are resistant to MEK and BRAF inhibitors, their response to immune checkpoint inhibitors is less robust than the response of BRAF mutant melanomas to these agents, and additional validated targets for therapeutic intervention have yet to be identified. Previous work from our laboratory has demonstrated that ERBB4 is required for the proliferation of the IPC-298, MEL-JUSO, MeWo, and SK-MEL-2 BRAF-WT&RAS/NF1-mutant melanoma cell lines. Surprisingly, the synthetic constitutively dimerized and active Q646C ERBB4 mutant allele appears to strongly inhibits the proliferation of BRAF-WT&RAS/NF1- mutant melanoma cell lines. Given that we have also previously demonstrated that ERBB4-ERBB2 and ERBB4-EGFR heterodimers are more potent drivers of proliferation than are ERBB4 homodimers, here we begin to test the hypothesis that ERBB4 heterodimers drive the proliferation of BRAF-WT&RAS/NF1-mutant melanoma cell lines. Here we demonstrate that the kinase-deficient (dominant-negative) ERBB2 K753A mutant allele inhibits the clonogenic proliferation of the IPC-298, MEL-JUSO, and MeWo ERBB4-dependent, BRAF-WT&RAS/NF1-mutant melanoma cell lines. Moreover, the kinase-deficient (dominant-negative) EGFR K721A mutant allele inhibits the clonogenic proliferation of the MeWo cell line, but not the IPC-298 or MEL-JUSO cell lines. Finally, the clonogenic proliferation of the SK-MEL-2 ERBB4-dependent, BRAF-WT&RAS/NF1-mutant melanoma cell line is unaffected by the ERBB2 K753A or EGFR K721A dominant-negative mutant alleles. We discuss these findings in the context of our hypothesis that ERBB4 heterodimers drive the proliferation of BRAF-WT&RAS/NF1-mutant melanoma cell lines.

cancer biology↗

bioRxiv2024

The Calmodulin-like proteins, CML13 and CML14 Function as Myosin Light Chains for the Class XI Myosins in Arabidopsis

Myosins are a crucial motor protein associated with the actin cytoskeleton in eukaryotic cells. Structurally, myosins form heteromeric complexes, with smaller light chains such as calmodulin (CaM) bound to isoleucine-glutamine (IQ) domains in the neck region. These interactions facilitate mechano-enzymatic activity. Recently, we identified Arabidopsis CaM-like (CML) proteins CML13 and CML14 as interactors with proteins containing multiple IQ domains, that function as the myosin VIII light chains. This study demonstrates that CaM, CML13, and CML14 specifically bind to the neck region of all 13 Arabidopsis myosin XI isoforms, with some preference among the CaM/CML-IQ domains. Additionally, we observed distinct residue preferences within the IQ domains for CML13, CML14, and CaM. In vitro experiments revealed that recombinant CaM, CML13, and CML14 exhibit calcium-independent binding to the IQ domains of myosin XIs. Furthermore, when co-expressed with MAP65-1-myosin fusion proteins containing the IQ domains of myosin XIs, CaM, CML13, and CML14 co-localize to microtubules. In vitro actin motility assays demonstrated that recombinant CML13, CML14, and CaM function as myosin XI light chains. A cml13 T-DNA mutant exhibited a shortened primary root phenotype that was complemented by the wild-type CML13 and was similar to that observed in a triple myosin XI mutant (xi3KO). Overall, our data indicate that Arabidopsis CML13 and CML14 are novel myosin XI light chains that likely participate in a breadth of myosin XI functions. HighlightMyosin XI proteins play a crucial role in the plant cytoskeleton, but their associated light chains have remained unidentified. Here, we show that calmodulin-like proteins, CML13 and CML14, serve as light chains for myosin XI, similar to their role for myosin VIII proteins

plant biology↗

bioRxiv2024

Mode of action of the toxic proline mimic azetidine 2-carboxylic acid in plants

Plants have an amazing capacity to outcompete neighboring organisms for space and resources. Toxic metabolites are major players in these interactions, which can have a broad range of effectiveness by targeting conserved molecular mechanisms, such as protein biosynthesis. However, lack of knowledge about defensive metabolite pathways, their modes of action, and resistance mechanisms limits our ability to manipulate these pathways for enhanced crop resilience. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites with a variety of functions but are typically not incorporated during protein biosynthesis. Here, we investigate the mode of action of the NPAA azetidine-2-carboxylic acid (Aze), an analog of the amino acid proline (Pro). Using a combination of plate-based assays, metabolite feeding, metabolomics, and proteomics, we show that Aze inhibits the root growth of Arabidopsis and other plants. Aze-induced growth reduction was restored by supplementing L-, but not D-Pro, and non-targeted proteomics confirms that Aze is misincorporated for Pro during protein biosynthesis, specifically on cytosolically translated proteins. qRT-PCR analysis and free amino acid profiling show that the unfolded protein response is upregulated during Aze treatment implicating protein degradation of misfolded proteins. This study demonstrates the mode of action of Aze in plants and provides a foundation for engineering Aze production and tolerance in crops for enhanced resilience.

plant biology↗

bioRxiv2024

Mitochondrial Transplantation promotes protective effector and memory CD4+ T cell response during Mycobacterium tuberculosis infection and diminishes exhaustion and senescence in elderly CD4+ T cells

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M.tb), remains a significant health concern worldwide, especially in populations with weakened or compromised immune systems, such as the elderly. Proper adaptive immune function, particularly a CD4+ T cell response, is central to host immunity against M.tb. Chronic infections, such as M.tb, as well as aging promote T cell exhaustion and senescence, which can impair immune control and promote progression to TB disease. Mitochondrial dysfunction contributes to T cell dysfunction, both in aging and chronic infections and diseases. Mitochondrial perturbations can disrupt cellular metabolism, enhance oxidative stress, and impair T-cell signaling and effector functions. This study examined the impact of mitochondrial transplantation (mito-transfer) on CD4+ T cell differentiation and function using aged mouse models and human CD4+ T cells from elderly individuals. Our study revealed that mito-transfer in naive CD4+ T cells promoted the generation of protective effector and memory CD4+ T cells during M.tb infection in mice. Further, mito-transfer enhanced the function of elderly human T cells by increasing their mitochondrial mass and modulating cytokine production, which in turn reduced exhaustion and senescence cell markers. Our results suggest that mito-transfer could be a novel strategy to reestablish aged CD4+ T cell function, potentially improving immune responses in the elderly and chronic TB patients, with a broader implication for other diseases where mitochondrial dysfunction is linked to T cell exhaustion and senescence.

immunology↗

bioRxiv2023

Functional Characterization of Calmodulin-like Proteins, CML13 and CML14, as Novel Light Chains of Arabidopsis Class VIII Myosins

Myosins are important motor proteins that associate with the actin cytoskeleton. Structurally, myosins function as heteromeric complexes where smaller light chains, such as calmodulin (CaM), bind to isoleucine-glutamine (IQ) domains in the neck regions to facilitate mechano-enzymatic activity. We recently identified Arabidopsis CaM-like (CML) proteins, CML13 and CML14 as interactors of proteins containing multiple IQ domains, including a member of the myosin VIII class. Here, using in vivo and in vitro assays we demonstrate that CaM, CML13, and CML14 bind the neck region of all four Arabidopsis myosin VIII isoforms. Among ten CML isoforms tested for in planta binding to myosins VIIIs, CaM, CML13, and CML14 gave the strongest signals using in planta split-luciferase protein-interaction assays. In vitro, recombinant CaM, CML13, and CML14 showed specific, high-affinity, calcium-independent binding to the IQ domains of myosin VIIIs. Subcellular localization analysis indicated that CaM, CML13, and CML14 co-localized to plasma membrane-bound puncta when co-expressed with RFP-myosin fusion proteins containing IQ- and tail-domains of myosin VIIIs. In addition, in vitro actin-motility assays using recombinant myosin holoenzymes demonstrated that CaM, CML13, and CML14 function as light chains for myosin VIIIs. Collectively, our data indicate that Arabidopsis CML13 and CML14 are novel myosin VIII light chains. HighlightMyosins are key proteins in the plant cytoskeleton, but the identity of their light chain components is unknown. Here, we show that calmodulin-like proteins function as novel myosin light chains.

plant biology↗

bioRxiv2023

Alleviating the barrier of adventitious roots formation in recalcitrant mature tissue by slow release of a synthetic auxin

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid (IBA) is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here, we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid-L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation.

plant biology↗

bioRxiv2022

Biochemical analysis of anthocyanin and proanthocyanidin and their regulation in determining chickpea flower and seed coat colours

Flower and seed coat colour are important agronomic traits in chickpea (Cicer arietinum L.). Cultivated chickpeas are of two types namely, desi (dark seeded, purple flower) and kabuli (light colour seeded, white flower). There has been limited information about the molecular mechanism underlying the colour variation of flower and seed coats in desi and kabuli chickpea. We profiled the anthocyanin and proanthocyanidin (PA) contents in chickpea flowers and seed coats. Tissue-specific silencing of two genes encoding a basic helix-loop-helix (CabHLH) protein and a tonoplast-localized multidrug and toxic compound extrusion (CaMATE1) transporter in a desi genotype resulted in the reduction in expressions of anthocyanin and PA biosynthetic genes and anthocyanin and PA contents in the flower and seed coat and produced flowers and seeds with kabuli characteristics. Transcriptional regulation of a subset of anthocyanin and PA biosynthetic genes by a natural CabHLH variant and transport assay of a natural CaMATE1 variant explained the association of these alleles with the kabuli phenotype. We carried out a detailed molecular characterization of these genes, and provided evidences that kabuli chickpea flower and seed colour phenotype can be derived by manipulation of single genes in a desi chickpea background. HighlightIn this study, we have defined the molecular link between flower and seed color in chickpea and identified CaMATE1 and CabHLH as the regulators of both the traits.

plant biology↗

bioRxiv2021

IL-10 receptor blockade delivered simultaneous with BCG vaccination sustains long term protection against Mycobacterium tuberculosis infection in mice

Mycobacterium bovis bacillus Calmette-Guerin (BCG) immunization still remains the best vaccination strategy available to control the development of active tuberculosis (TB). Protection afforded by BCG vaccination gradually wanes over time and while booster strategies have promise, they remain under development. An alternative approach is to improve BCG efficacy through host-directed therapy. Building upon prior knowledge that blockade of interleukin-10 receptor 1 (IL-10R1) during early Mycobacterium tuberculosis (M.tb) infection improves and extends control of M.tb infection in mice, we employed a combined anti-IL-10R1/BCG vaccine strategy. A subcutaneous, single vaccination of BCG/IL10-R1 increased the numbers of CD4+ and CD8+ central memory T cells, and reduced TH1 and TH17 cytokine levels in the lung for up to 7 weeks post vaccination. Subsequent M.tb challenge in mice showed both an early (4 week) and sustained long-term (47 week) control of infection, which was associated with increased survival. In contrast, protection of BCG/saline vaccinated mice waned 8 weeks post M.tb infection. Our findings demonstrate that a single and simultaneous vaccination with BCG/IL10-R1 sustains long-term protection, identifying a promising approach to enhance and extend the current BCG mediated protection against TB.

immunology↗

bioRxiv2021

Rapid and Efficient Inactivation of SARS-CoV-2 from Surfaces using UVC Light Emitting Diode Device

Efforts are underway to develop countermeasures to prevent the environmental spread of COVID-19 pandemic caused by SARS-CoV-2. Physical decontamination methods like Ultraviolet radiation has shown to be promising. Here, we describe a novel device emitting ultraviolet C radiation (UVC), called NuvaWave, to rapidly and efficiently inactivate SARS-CoV-2. SARS-CoV-2 was dried on a chambered glass slides and introduced in a NuvaWave robotic testing unit. The robot simulated waving NuvaWave over the virus at a pre-determined UVC radiation dose of 1, 2, 4 and 8 seconds. Post-UVC exposure, virus was recovered and titered by plaque assay in Vero E6 cells. We observed that relative control (no UVC exposure), exposure of the virus to UVC for one or two seconds resulted in a >2.9 and 3.8 log10 reduction in viral titers, respectively. Exposure of the virus to UVC for four or eight seconds resulted in a reduction of greater than 4.7-log10 reduction in viral titers. The NuvaWave device inactivates SARS-CoV-2 on surfaces to below the limit of detection within one to four seconds of UVC irradiation. This device can be deployed to rapidly disinfect surfaces from SARS-CoV-2, and to assist in mitigating its spread in a variety of settings.

microbiology↗

bioRxiv2021

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient (upf1{Delta}) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1{Delta} cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1{Delta}. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

molecular biology↗

bioRxiv2021

Extracellular Delivery of Functional Mitochondria Reverses the Dysfunction of CD4+ T Cells in Aging

Mitochondrial dysfunction alters cellular metabolism, increases tissue oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, we investigated whether the transfer of functional mitochondria into CD4+ T cells that were isolated from old mice (aged CD4+ T cells), could abrogate aging-associated mitochondrial dysfunction, and improve the aged CD4+ T cell functionality. Our results show that the delivery of exogenous mitochondria to aged non-activated CD4+ T cells led to significant mitochondrial proteome alterations highlighted by improved aerobic metabolism and decreased cellular mitoROS. Additionally, mito-transferred aged CD4+ T cells showed improvements in activation-induced TCR-signaling kinetics displaying markers of activation (CD25), increased IL-2 production, enhanced proliferation ex vivo. Importantly, immune deficient mouse models (RAG-KO) showed that adoptive transfer of mito-transferred naive aged CD4+ T cells, protected recipient mice from influenza A and Mycobacterium tuberculosis infections. These findings support mitochondria as targets of therapeutic intervention in aging.

immunology↗

bioRxiv2020

Lethality of SARS-CoV-2 infection in K18 human angiotensin converting enzyme 2 transgenic mice

ABSTRACTVaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

microbiology↗