Biology subjects

Kumar, R.

Publications and source records attributed to Kumar, R..

At least 109 records · Page 6Linked to original sources

bioRxiv2025

Gut sulfide metabolism modulates behavior and brain bioenergetics

The host-microbiome interface is rich in metabolite exchanges and exquisitely sensitive to diet. Hydrogen sulfide (H2S) is present at high concentrations at this interface, and is a product of both microbial and host metabolism. The mitochondrial enzyme, sulfide quinone oxidoreductase (SQOR), couples H2S detoxification to oxidative phosphorylation; its inherited deficiency presents as Leigh disease. Since an estimated two thirds of systemic H2S metabolism originates in gut, it raises questions as to whether impaired sulfide clearance in this compartment contributes to disease, and whether it can be modulated by dietary sulfur content. In this study, we report that SQOR deficiency confined to murine intestinal epithelial cells, perturbs colon bioenergetics that is reversed by antibiotics, establishing a significant local contribution of microbial H2S to host physiology. We also find that a 2.5-fold higher methionine intake, mimicking the difference between animal and plant proteins, synergized with intestinal SQOR deficiency to adversely impact colon architecture and alter microbiome composition. In serum, increased thiosulfate, a biomarker of H2S oxidation, revealed that intestinal SQOR deficiency combined with high dietary methionine, affects sulfide metabolism globally and perturbs energy metabolism as indicated by higher ketone bodies. The mice exhibited lower exploratory locomotor activity while brain MRI revealed an atypical reduction in ventricular volume, which was associated with lower aquaporin 1 that is important for cerebrospinal fluid secretion. Our study reveals the dynamic interaction between dietary sulfur intake and sulfide metabolism at the host-microbe interface, impacting gut health, and the potential for lower dietary methionine intake to modulate pathology. Significance StatementThe host-microbiome interface is rich in metabolite-based communications that are modulated by diet. Hydrogen sulfide (H2S), which is a respiratory poison at high concentrations, is enriched at this interface, and is detoxified by the host enzyme, sulfide quinone oxidoreductase (SQOR). Given the quantitatively significant contribution of gut to systemic H2S metabolism, we examined how SQOR deficiency restricted to murine intestinal epithelial cells, interacts with high dietary methionine, designed to approximate the difference between plant versus animal protein levels, to affect local and global bioenergetics. Our study revealed profound short- and long-range impacts resulting from the synergy between decreased H2S clearance capacity in gut and high dietary methionine on global energy metabolism, brain pathology, and behavior.

biochemistry↗

bioRxiv2025

TMT-based quantitative proteomic assessment of Vicia sativa induced neurotoxicity by β-cyano-L-alanine and γ-glutamyl-β-cyano-L-alanine in SH-SY5Y cells.

{beta}-Cyano-L-alanine (BCA) and {gamma}-glutamyl-{beta}-cyano-L-alanine (GBCA) are the primary antinutritional compounds present within the seeds of the high protein and drought tolerant orphan legume Vicia sativa. Evidence of neurotoxicity is limited to symptom analysis from animal feed trials whilst the molecular mechanisms underlying suspected neurotoxicity in monogastric animals are largely unknown. In this study, we first optimised an in vitro cell-based assay for rapid testing of BCA and GBCA toxicity in the retinoic acid differentiated SH-SY5Y human neuroblastoma cells. Using this system, we then performed proteomics analyses to determine dysregulated expression of proteins in BCA or GBCA treated of differentiated SH-SY5Y cells. Our findings indicate that BCA affects expression of proteins involved in DNA damage and translation whilst GBCA treatment causes dysregulation of those involved in mitosis and cell cycle. Following BCA treatment, we identified changes in many proteins previously suggested to have close association with neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimers disease as well as cancers. Following GBCA treatment, dysregulation of proteins involved in apoptosis pathways was observed. Finally, the lack of common dysregulated proteins and pathways in BCA or GBCA treated cells indicated that they most likely cause neurotoxicity via distinct mechanisms. SignificanceAntinutritional compounds or toxins limit the use of many potential grain crops including V. sativa (Common Vetch), and BCA and GBCA are the two principal toxic compounds in Common Vetch grain. Vetch grain toxicity is associated with neurotoxic symptoms in animals. Characterizing the proteome of BCA- and GBCA-treated neural cells gives insights into the excitotoxicity mechanism and identification of biomarkers for screening higher quality grain. Quantitative tandem mass tag (TMT) mass spectrometry-based proteome profiling of BCA treated neural cells characterized 6,827 proteins, of which 26 were significantly up-regulated and 73 significantly downregulated. However, TMT proteome analysis of GBCA treated cells identified 76 significantly up regulated and 86 downregulated proteins. These results provide insight into the toxicity of BCA and GBCA and enable future identification of putative direct protein targets. We describe for the first time BCA playing a role in modulating the expression DNA damage proteins and translation, and GBCA playing a role in modulating the expression of mitosis and cell cycle proteins, elucidating the mechanisms of plant-derived toxins in mammalian cell neurotoxicity HighlightsO_LIEstablished in vitro cell-based toxicity assay for {beta}-cyano-L-alanine and {gamma}-glutamyl-{beta}-cyano-L-alanine. C_LIO_LI{beta}-Cyano-L-alanine treatment dysregulated many proteins associated with DNA damage in retinoic acid differentiated SH-SY5Y cells. C_LIO_LI{gamma}-Glutamyl-{beta}-cyano-L-alanine treatment dysregulated proteins associated with cell cycle. C_LI

pharmacology and toxicology↗

bioRxiv2025

High Density Phenotypic Map of Natural Variation for Intermediate Phenotypes Associated with Stalk Lodging Resistance in Maize

The world has food security needs that are currently not being met. Stalk lodging undermines crop productivity and incurs global yield losses of at least $6 billion in maize (Zea mays L.). Genetic architecture of stalk lodging resistance, a measure of the ability of the stalk to withstand lodging, remains poorly resolved, creating a bottleneck for genetic improvement. Identification of diverse plant traits at multiple length scales of biological organizations that contribute to stalk lodging resistance and characterization of natural variation for these traits is critical for improving stalk lodging resistance. We identified and evaluated 11 intermediate phenotypes, traits associated with stalk lodging resistance, in a maize diversity panel of 566 inbred lines evaluated over four environments. The identity of each of the 31,260 stalks evaluated in the study was preserved throughout the phenotyping pipeline which enabled capturing variation at the individual plant level. This high-density phenotypic dataset provided a foundation for statistical genomics, predictive modeling, and machine learning analyses to identify genes and genetic elements underlying stalk lodging resistance. Additionally, phenotypic characterization of multiple intermediate phenotypes on a diverse set of inbred lines provided excellent opportunities to understand the relative contribution of these traits to stalk lodging resistance. Besides improvement of maize for grain and animal feedstock, the inferences from this data will be valuable for improvement of stalk lodging resistance in other grass species.

plant biology↗

bioRxiv2025

Human sensory neurons exhibit cell-type-specific, pain-associated differences in intrinsic excitability and expression of SCN9A and SCN10A

Despite major advances in pain science, the approval of novel therapeutics has been slow. A major cause for the lack of new analgesics may be fundamental biological differences between humans and model organisms used in preclinical research. Large-scale transcriptional profiling efforts on human dorsal root ganglia (hDRG) have now identified at least 22 distinct neuronal subtypes; however, a significant knowledge gap exists in ascribing functional phenotypes to these diverse neuronal populations. In this study, we use Patch-seq recordings in hDRG to link electrical properties to transcriptionally defined cell types. First, through unbiased clustering of electrophysiological properties from 228 hDRG neurons, we identify three electrophysiological subtypes (E-types). Next, we show that E-types can be mapped onto specific transcriptional classes (T-types) of hDRG neurons. We find that donors pain history is associated with E-type-specific differences in electrical properties, some of which may be associated with higher expression of voltage-gated sodium channels, NaV1.7 and NaV1.8. These results highlight the importance of using multimodal profiling to better understand human sensory neuron biology and may help reveal novel therapeutic targets driving chronic pain. TeaserMapping electrical properties to transcriptional profiles of human sensory neurons offers new insights into pain neurobiology

neuroscience↗

bioRxiv2025

TGF-β inhibitor SB431542 suppresses coronavirus replication through multistep inhibition

The COVID-19 pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-{beta} receptor I (ALK5) inhibitor, exhibits potent antiviral activity against SARS-CoV-2 through unprecedented multitargeted mechanisms. Through comprehensive in vitro, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupt its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36-48 hpi), SARS-CoV-2 exploits TGF-{beta}-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release--processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC50 of 515 nM against SARS-CoV-2. In vivo efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favourable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-{beta}/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-{beta}-induced apoptosis during late-stage infection-- collectively imposing multiple selective constraints that impede escape mutation development. ImportanceThe COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-{beta} signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure--a significant advantage over current therapeutics that quickly lose effectiveness due to viral mutations. Our findings also uncover that coronaviruses exploit both lysosomal dysfunction and programmed cell death to spread efficiently, providing new targets for therapeutic intervention. This research establishes SB431542 as a promising broad-spectrum coronavirus inhibitor and demonstrates the value of targeting host-virus interactions to overcome antiviral resistance.

microbiology↗

bioRxiv2025

Alternative Promoters Drive Transcriptomic Reprogramming and Prognostic Stratification in TNBC

Transcriptional regulation frequently involves alternative promoters, yet the distinct regulatory mechanisms governing alternative versus reference promoters remain poorly understood in Triple-Negative Breast Cancer (TNBC), a high-risk breast tumor subtype. It is worth emphasizing that, despite the availability of extensive short-read sequencing data, the impact of alternative promoter usage on TNBC transcriptome dynamics and patient survival remains underexplored. The current study leverages RNA sequencing data from the publicly available TNBC tumor samples (360) and adjacent normal samples (88) to identify TNBC-specific and subtype-specific active alternative promoters (AAPs). To further validate these findings, we integrated H3K4me3 ChIP-seq data, confirming key promoter switching events. We found that HDAC9, RPS14, and EPN1 exhibit tumor-specific AAP expression, while AKAP9 and SEC31A show basal subtype-specific promoter activity in TNBC. Beyond their transcriptional impact, we also explored the prognostic significance of AAPs. The alternative promoters of HUWE1 and FTX were recognized as independent survival predictors in TNBC. Notably, multivariate analysis demonstrated that prognostic AAPs remained significant in predicting relapse-free survival (RFS) even after adjusting for copy number alterations (CNA) and mRNA-based subtypes. Our AAP-based prognostic model achieved C-index of 0.73 in training and 0.72 in validation, with AUROC of 0.72 and integrated Brier score of 0.09 in validation dataset. Our findings indicate that AAP activity serves as a crucial prognostic marker beyond traditional clinical parameters, enhancing patient stratification and risk assessment in TNBC. Understanding promoter switching events may further unveil novel therapeutic targets, paving the way for precision oncology strategies in highly aggressive breast tumors.

genomics↗

bioRxiv2025

Multi-omic landscape of human gliomas from diagnosis to treatment and recurrence

Gliomas are among the most lethal cancers, with limited treatment options. To uncover hallmarks of therapeutic escape and tumor microenvironment (TME) evolution, we applied spatial proteomics, transcriptomics, and glycomics to 670 lesions from 310 adult and pediatric patients. Single-cell analysis shows high B7H3+ tumor cell prevalence in glioblastoma (GBM) and pleomorphic xanthoastrocytoma (PXA), while most gliomas, including pediatric cases, express targetable tumor antigens in less than 50% of tumor cells, potentially explaining trial failures. Longitudinal samples of isocitrate dehydrogenase (IDH)-mutant gliomas reveal recurrence driven by tumor-immune spatial reorganization, shifting from T-cell and vasculature-associated myeloid cell-enriched niches to microglia and CD206+ macrophage-dominated tumors. Multi-omic integration identified N-glycosylation as the best classifier of grade, while the immune transcriptome best predicted GBM survival. Provided as a community resource, this study opens new avenues for glioma targeting, classification, outcome prediction, and a baseline of TME composition across all stages.

cancer biology↗

bioRxiv2025

Temporal analysis of physiological phenotypes identifies novel metabolic and genetic underpinnings of senescence in maize

Leaf senescence induces extensive metabolome reprogramming to optimize nutrient recycling, enhance resilience to abiotic and biotic stress, and improve productivity. However, the characterization of these metabolic shifts and the identification of key metabolites and pathways remains limited. We generated a temporal map of physiological and metabolic diversity in genetically diverse maize inbred lines varying for the staygreen trait. Combinatorial analysis of physiological and metabolic changes revealed substantial metabolic perturbations and identified 84 leaf metabolites associated with senescence. Non-staygreen inbred lines exhibited higher accumulation of primary metabolites including sugar alcohols such as mannitol and erythritol, and amino acids such as phenylalanine and arginine. In contrast, the staygreen inbred lines showed higher abundance of secondary metabolites, primarily phenylpropanoids, including caffeic acid, chlorogenic acid, and eriodictyol. Linking metabolome to the genome identified 56 novel candidate genes expressed in adult maize leaf that regulate metabolic flux during senescence. Reverse genetic analysis validated the role of naringenin chalcone and eriodictyol in both maize and Arabidopsis, demonstrating a conserved function of these phenylpropanoids in leaf senescence across monocots and dicots. Our study provides valuable insights into the coordinated physiological and metabolic changes driving leaf senescence and identifies novel genes underlying this complex developmental process.

plant biology↗

bioRxiv2025

Impact of Long-Term Integrated Nutrient Management Practices on Soil Bacterial Diversity: A 16S rRNA-Based Metagenomic Approach

The growing worlds population, expected to surpass 9 billion by 2050, demands a 70-100% increase in farm productivity, posing significant challenges for sustainable agriculture. Conventional farming, reliant on chemical fertilizers and pesticides, negatively impacts environmental sustainability and food quality. Metagenomics, a culture-independent technique, has revolutionized the study of microbial communities by enabling comprehensive analysis of microbial diversity and function. This study evaluated the effects of combined nutrient management practices--including farmyard manure (FYM), wheat straw, green manure, and NPK fertilizers--on soil bacterial diversity using 16S rRNA-based metagenomic analysis. Key findings revealed the dominance of bacterial phyla such as Proteobacteria, Actinobacteria, and Firmicutes, which play critical roles in nutrient cycling, organic matter decomposition, and plant growth promotion. Distinct taxonomic distributions were observed across the samples, with Proteobacteria and Firmicutes dominating in control (T1), NPK (T2), and NPK_Green Manure (T5) treatments, while a shift in phyla composition was noted in NPK_FYM (T3) and NPK_WheatStraw (T4). Genus-level analysis revealed that Pseudomonas and Bacillus species were abundant in control (T1) and NPK (T2), while Sphingomonas species were dominant in NPK_FYM (T3). Further diversity analyses revealed significant variation across the samples, with T3 showing the highest richness and control (T1) showing the lowest richness. The rarefaction curve confirmed sufficient sampling for capturing microbial diversity, while beta diversity analysis, including Principal Coordinate Analysis (PCA) and Bray-Curtis clustering, indicated distinct microbial community structures across different treatments. The combined use of organic amendments with NPK fertilizer enhanced microbial diversity and population, indicating their potential to support resilient and productive soil ecosystems.

bioinformatics↗

bioRxiv2025

A screen to identify antifungal antagonists reveals a variety of pharmacotherapies induce echinocandin tolerance in Candida albicans.

Through screening a comprehensive collection of drugs approved for human use, we identified over 20 that oppose the antifungal activity of the echinocandins upon the infectious yeast, Candida albicans. More detailed evaluation of five such drugs, including the atypical antipsychotic aripiprazole and the tyrosine kinase inhibitor ponatinib, indicated they promote C. albicans survival following exposure to the echinocandin antifungals. The activity of the five selected antagonists was dependent upon the Mkc1p MAPK pathway, however, ponatinib was paradoxically shown to suppress phosphorylation and therefore activation of Mkc1p itself. Components of several other signaling pathways are also required, including those of calcineurin and casein kinase-2, suggesting the observed antagonism required much of the cell wall stress responses previously described for C. albicans. Transcriptome analysis revealed that the antagonists stimulated the expression of genes involved in xenobiotic and antifungal resistance, and suppressed the expression of genes associated with hyphal growth. Thus, the echinocandin antagonistic drugs modulate C. albicans physiology in ways that could impact its pathogenicity and/or response to therapeutic intervention. Finally, a mutant lacking the Efg1p transcription factor, which has a central role in the activation of C. albicans hyphal growth was found to have intrinsically high levels of echinocandin tolerance, suggesting a link between modulation of morphogenesis related signaling and echinocandin tolerance. ImportanceWe report a substantial number of previously unknown drug interactions that modulate the echinocandin sensitivity of one of the most prevalent human fungal pathogens, Candida albicans. The echinocandins are the first line therapy for treating disseminated and often lethal Candida infections, that account for >75% of invasive fungal infections in the U.S.. For largely unknown reasons, a substantial number of patients with invasive candidiasis fail to respond to treatment with these drugs. The finding of this study suggest that co-administered medications have the potential to influence the therapeutic outcomes of invasive fungal infections through modulating antifungal drug tolerance and/or fungal pathogenicity. The potential for echinocandin antagonistic medications to influence therapeutic outcomes is discussed.

microbiology↗

bioRxiv2025

Estimating physical conditions supporting gradients of ATP concentration in the eukaryotic cell

The ATP molecule serves as an energy currency in eukaryotes (and all life), providing the energy needed for many essential cellular processes. But the extent to which substantial spatial differences exist in ATP concentration in the cell remains incompletely known. It is variously argued that ATP diffuses too quickly for large gradients to be established, or that the high rates of ATP production and use (sources and sinks) can support large gradients even with rapid diffusion - and microscopic models and detailed experiments in different specific cases support both pictures. Here we attempt a mesoscopic investigation, using reaction-diffusion modelling in a simple biophysical picture of the cell to attempt to ask, generally, which conditions cause substantial ATP gradients to emerge within eukaryotic cells. If ATP sources (like mitochondria) or sinks (like the nucleus) are spatially clustered, large fold changes in concentration can exist across the cell; if sources and sinks are more spread then rapid diffusion indeed prevents large gradients being established. This dependence holds in model cells of different sizes, suggesting its generality across cell types. Our theoretical work will complement developing intracellular approaches exploring ATP concentration inside eukaryotic cells.

cell biology↗

bioRxiv2025

Heterogeneous T cell response patterns within established clinical phases of Hepatitis B infection revealed through the rapid functional characterization of HBV-specific T cells in whole blood

Background & AimsThere is an unmet need for immunological biomarkers in chronic hepatitis B (CHB), where patient management relies on virological and biochemical markers despite the crucial role of virus-specific T cells in controlling viral replication and disease progression. To address this, we developed the HBV-cytokine release assay (HBV-CRA), a rapid, point-of-care test that measures multiple cytokines in whole blood after HBV-peptide stimulation. MethodsWe first assessed the assays sensitivity by spiking whole blood with engineered HBV-specific T cells. Next, we compared sensitivity and consistency of HBV-CRA to ex vivo IFN-{gamma} ELISpot assays. We then applied the assay in a cross-sectional study of 235 CHB patients and longitudinally in acute HBV patients during HBsAg sero-clearance. ResultsThe HBV-CRA detected T cell function in 80% of CHB cases and showed that elevated IL-2 and IFN-{gamma} levels after Core peptide stimulation were associated with HBsAg clearance. Importantly, unsupervised clustering identified distinct immune response patterns independent of established clinical and virological classifications. The assay also demonstrated the functional impact of NUC treatment on HBV-specific T cell responses. ConclusionsThe HBV-CRA is a rapid and easy-to-use assay that identifies immune profiles associated with HBsAg clearance and differentiates CHB patients based on antiviral T cell function. Its application in a large CHB cohort revealed that traditional disease phase classifications, based on viral and clinical parameters, cannot predict HBV-specific T cell profiles. The HBV-CRA has the potential to guide patient stratification for immunotherapeutic interventions.

immunology↗

bioRxiv2025

Dynamic Reprogramming of Stromal Pdgfra-expressing cells during WNT-Mediated Transformation of the Intestinal Epithelium

Stromal fibroblasts regulate critical signaling gradients along the intestinal crypt-villus axis1 and provide a niche that supports adjacent epithelial stem cells. Here we report that Pdgfra-expressing fibroblasts secrete ligands that promote a regenerative-like state in the intestinal mucosa during early WNT-mediated tumorigenesis. Using a mouse model of WNT-driven oncogenesis and single-cell RNA sequencing (RNA-seq) of mesenchyme cell populations, we revealed a dynamic reprogramming of Pdgfra+ fibroblasts that facilitates WNT-mediated tissue transformation. Functional assays of potential mediators of cell-to-cell communication between these fibroblasts and the oncogenic epithelium revealed that TGFB signaling is notably induced in Pdgfra+ fibroblasts in the presence of oncogenic epithelium, and TGFB was essential to sustain regenerative-like growth of organoids ex vivo. Genetic reduction of Cdx2 in the {beta}-catenin mutant epithelium elevated the fetal-like/regenerative transcriptome and accelerated WNT-dependent onset of oncogenic transformation of the tissue in vivo. These results demonstrate that Pdgfra+ fibroblasts are activated during WNT-driven oncogenesis to promote a regenerative state in the epithelium that precedes and facilitates formation of tumors.

cancer biology↗

bioRxiv2025

Small molecule promoters of endogenous lipid droplet accumulation drive lysophagy

Lipid droplets (LDs) play a central role in regulating metabolism in stress-induced conditions, including one triggered by nutrient deprivation. Unravelling the protein networks involved in the biogenesis of LDs and their causative and functional roles in health and disease continue to evolve. To this cause, genetic manipulation of the lipid metabolic network or supplementation of high fat diet/ oleic acid (OA) are the traditional routes for voluntarily triggering LDs formation in cells and animals. We developed a screening platform for the identification of new LDs inducers, and our primary screening of various fatty acids identified linoleic acid (LOA, DUFA) as a better tool than OA (MUFA) in promoting LDs formation. The screening and validation discovered new small molecule-based tools for promoting a rapid organization of endogenous lipids into droplets in multiple cell types. Notably, our mass spectral lipidomics analysis presented the overproduction of phosphatidylcholines and small triglycerides, a hallmark of LDs. Mechanistic investigations of our lead molecules highlighted lipid peroxidation and ATP depletion through mitochondrial impairment in cells, which could serve as chemical cues for driving the fusion of cellular lipids into LDs. Finally, we uncovered the abrupt levels of LDs formation induced by our molecules promoted lysophagy in cancer cells to prevent their proliferation. Collectively, our work introduces new small molecules as powerful tools for reliably promoting LDs accumulation for studying their roles in biology, and we demonstrate the over accumulation of LDs prevent cancer cell proliferation, movement, and colonization.

cancer biology↗

bioRxiv2025

Virus and Cell Specific HMGB1 Secretion and Subepithelial Infiltrate Formation in Adenovirus Keratitis

A highly contagious infection caused by human adenovirus species D (HAdV-D), epidemic keratoconjunctivitis (EKC) results in corneal subepithelial infiltration (SEI) by leukocytes, the hallmark of the infection. To date, the pathogenesis of corneal SEI formation in EKC is unresolved. HMGB1 (high-mobility group box 1 protein) is an alarmin expressed in response to infection and a marker of sepsis. Earlier studies using a different adenovirus species, HAdV-C, showed retention of HMGB1 in the infected cell nucleus by adenovirus protein VII, enabling immune evasion. Here, using HAdV-D we show cell-specific HMGB1 secretion by infected cells, and provide an HAdV-D specific mechanism for SEI formation in EKC. HMGB1 was secreted only upon infection of human corneal epithelial cells, not from other cell types, and only upon infection by HAdV-D types associated with EKC. Acetylated HMGB1 translocation from the nucleus to the cytoplasm, then to the extracellular milieu, was tightly controlled by CRM1 and LAMP1, respectively. Primary stromal cells when stimulated by rHMGB1 expressed proinflammatory chemokines. In a novel 3D culture system in tune with the architecture of the cornea, HMGB1 released by infected corneal epithelial cells induced leukocytic infiltrates either directly and/or indirectly via stimulated stromal cells, which together explains SEI formation in EKC.

microbiology↗

bioRxiv2024

Discovery and Biosynthesis of Nyuzenamides D and E by Genome Mining in Streptomyces hygroscopicus

Nyuzenamides belong to the class of bioactive cinnamoyl moiety containing non-ribosomal peptides (NRPs). However, their biosynthetic gene cluster (BGC) remains unconfirmed. Genome-mining revealed a putative nyu BGC in Streptomyces hygroscopicus DSM 40578. Nyuzenamides D (1) and E (2) were subsequently isolated, and the structures were elucidated by detailed NMR spectroscopic and MS spectrometric data analyses. The absolute configuration of 1 was determined by a single-crystal X-ray diffraction study. Through retro-biosynthesis and CRISPR-genome editing, the non-ribosomal peptide synthetase biosynthetic gene cluster for nyuzenamides was confirmed. Our discovery enriches the diversity of cinnamoyl-containing nonribosomal peptides and validates the biosynthetic gene clusters for future genome-mining research.

microbiology↗

bioRxiv2024

From Pigments to Precision: Exploring Genetic Transformation and Genome Editing in Wheat and Barley

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system, that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we primarily generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knockingout the first betalain biosynthetic gene in RUBY-positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods.

genomics↗

bioRxiv2024

Modelling benefits and costs of decision making and feedback control for organisms in changing environments

Cells (and organisms) make decisions in response to their environments. These decisions may help organism survival in environments with limited resource, but also constitute a cost to the organism in terms of the energy involved in sensing, processing, and responding to environmental change. Here, we explore the tradeoffs involved in a cost-benefit analysis of model organisms facing challenging deterministic or stochastic environments. The benefits of tunable versions of proportional-integral-derivative (PID) control are computed under different environmental behaviours; the model reflects both the ability to use this control to decide on cellular strategy and the potential cost associated with this feedback control. We quantify the circumstances under which control is most and least beneficial and the different weightings of the PID terms that perform best in specific and general situations. While our model is very simple, these results provide potential insight into the benefits of different control mechanisms, particularly at the single-cell level.

systems biology↗