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bioRxiv2022

Single-nucleotide Differences and Cell Type Decide the Subcellular Localization of miRNA Isoforms (isomiRs), tRNA-derived Fragments (tRFs) and rRNA-derived Fragments (rRFs)

BackgroundMicroRNAs (miRNAs) and their isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) represent ~95% of all short RNAs found in cells. All three types modulate mRNA and protein abundance and are dysregulated in diseases. Experimental studies to date assumed that the subcellular localization of these molecules is well understood and constant across cell types. ResultsWe investigated the localization of isomiRs, tRFs, and rRFs in biological replicates from three frequently-used model cell lines. In each case, we analyzed the contents of the nucleus, cytoplasm, whole mitochondrion, mitoplast, and the whole cell. We used a rigorous mathematical model to account for cross-fraction contamination and technical errors and adjusted abundances accordingly. We found that isomiRs, tRFs, and rRFs exhibit complex and unexpected patterns of subcellular localization. These patterns depend on the type of the RNA molecule, its exact sequence, and the cell type. Even for "sibling" RNAs from the same parental RNA whose sequences differ by only a few nucleotides, their subcellular localization depends on each siblings exact sequence and the cell type. ConclusionsPrevious studies of isomiRs, tRFs, and rRFs that used ectopic expression without accounting for isoforms may need to be re-evaluated. Future experiments with these molecules will need to distinguish among the multiple isoforms and account for the fact that each isoforms abundance and destination depend on its exact sequence and cell type. The findings additionally suggest the existence of an intracellular trafficking program for isomiRs, tRFs, and rRFs and, by extension, expanded roles for these molecules - both dimensions await characterization. To help design future experiments, we compiled a first-of-its-kind Atlas to catalogue the subcellular localization and abundance of 5,898 isomiRs, tRFs, and rRFs across three model cell lines. Results SummaryO_LIWe analyzed the distribution of microRNA isoforms (isomiRs), tRNA-derived fragments (tRFs), and rRNA-derived fragments (rRFs) in the{whitebullet} nucleus {whitebullet}cytoplasm {whitebullet}mitochondrion, and {whitebullet}mitoplast of biological replicates from three cell lines from the same tissue. C_LIO_LIWe corrected the measured abundances by accounting for cross-fraction contamination and technical errors through a rigorous mathematical model. C_LIO_LIOur analysis revealed complex localization patterns involving numerous isomiRs, tRFs, and rRFs. C_LIO_LIThe subcellular localization of these RNAs depends on their exact sequence and differs even for molecules that arise from the same parental miRNA, tRNA, or rRNA. C_LIO_LIFor a given RNA, its subcellular localization additionally depends on cell type. C_LIO_LIThe findings have implications for previous and future molecular studies of the function of isomiRs, tRFs, and rRFs. C_LIO_LIThe findings suggest the existence of a complex subcellular trafficking program, and hint at expanded functions for these RNA molecules that differ by compartment. C_LIO_LITo assist with the design of future experiments, we created a first-of-its-kind Atlas that catalogues the subcellular distribution and abundance of 5,898 isomiRs, tRFs, and rRFs across three cell lines. C_LI

molecular biology↗

bioRxiv2023

Identification roles of NFE2L3 in Digestive System Cancers

(1) BackgroundThe morbidity and mortality rate of Digestive System Cancers (DSC) continue to threaten human lives and health. Nuclear factor erythroid 2-like protein 3 (NFE2L3) is associated with the development, growth, and progression of multiple cancers. Nevertheless, the clinical value and underlying mechanisms of NFE2L3 in DSC remains unknown. This study aimed to clarify the possibilities of NFE2L3 as a novel biomarker in DSC. (2) MethodsWe obtained the data of NFE2L3 in cancers from database to evaluate the expression level and clinical value of NFE2L3 in DSC, and to explore underlying mechanism and biological functions of NFE2L3 in DSC. (3) ResultsNFE2L3 expression is up-regulated in DSC and have both prognostic and diagnostic value. NFE2L3 contributes to oncogenesis through a variety of mechanisms. In vitro experiment showed that NFE2L3 stimulated the proliferation and migration ability of gastric cancer cells. (4) ConclusionOur study confirms the clinical applicability of NFE2L3 as a promising biomarker for DSC.

molecular biology↗

bioRxiv2025

The coordinated regulatory roles of two LysR-Type Transcriptional Regulators balance chorismate and protocatechuate partition in Listeria organisms

Listeria monocytogenes is an economically deleterious foodborne pathogens that continually challenges the global food supply chain. Listeria species in general, synthesize protocatechuate from saprophytically-derived quinate and shikimate utilizing a novel class of bacterial dehydroshikimate dehydratase. Paradoxically, Listeria species are unable to metabolically utilize protocatechuate, as such, it was proposed that this compound is used as a currency to Listeria interactions with other microorganisms to improve their environmental proliferation. Therefore, an understanding of the regulatory mechanism for the metabolic pathway for protocatechuate biosynthesis is of great importance. Two LysR Type Transcriptional Regulators (LTTR), annotated QuiR and in this study QuiR2, are found upstream of genomic operons, qui1 and qui2, which transcribe genes for protocatechuate synthesis. QuiR, has been shown to activate the expression of genes from both operons with shikimate as a coinducer. However, the role of QuiR2, Lmo2233, is not clear. In this study, we conducted structural, biochemical and bioinformatics analyses of QuiR2 and demonstrated that it functions as a negative regulator of protocatechuate biosynthesis in Listeria species. Moreover, we determined that protocatechuate functions in modulating QuiR2 repressive properties through our mobility shift assay and LacZ reporter activity studies. Furthermore, phylogenetic analyses reveal that QuiR2 clusters closely but independently from QuiR thus supporting their distinct regulatory roles. We propose that QuiR2 prevents metabolic commitment of dehydroshikimate to protocatechuate when elevated and in limiting shikimate condition. In this study we revisited the biological role of the shikimate pathway in microbes and demonstrated that in addition to it producing chorismite for aromatic compound metabolism it is also important in allowing organisms to shuttle shikimate and quinate to produce protocatechuate which can be used as an energy source and more importantly in Listeria it is used to facilitate microbial interactions.

molecular biology↗

bioRxiv2025

Polysaccharides in cryopreservation: multidimensional systematic review of extremophilic traits and the role of selective pressure in structure-function relationships

Cryopreservation of biological matter has accumulated apex biomedical interest for its potential in elongating the shelf-life of biological matter in a state of suspended animation. In Nature, extremophilic microorganisms have an outstanding ability of surviving in habitats where extreme cold, heat, salinity and acidity defy the established boundaries for life. Through Darwinian selective adaptation, they have developed biochemical defense strategies to counteract lethal stimuli. Here, we have compiled an extremophilic EPS structure-function relationship database (XPOL-DB) which aggregates reports on 145 extremophilic and mesophilic EPS, for a total of 128 biochemical and establishes the psychrophilic chemical profile for cold adaptation. Psychrophilic EPS are highly-branched, polyanionic, elongated structures of increased flexibility and molecular weight (16-300 MDa), with predominant expression of polar monomers (GalNAc, GalA, GlcNAc, GlcA) - compared to the linear, rigid, neutral thermophilic EPS. This critical analysis revealed the significant EPS similarity between psychrophiles and halophiles suggests ice growth and extreme salinity are rooted in a shared mechanism of physical membrane destabilization, for which similar chemical traits can dualistically imbue freeze and salt tolerance. Psychrophiles are exciting testbeds for the mapping of how extreme cold selects for cryobiological EPS adaptation; and can fuel reverse engineering efforts to design optimal bio-based, non-cytotoxic cryoprotectant polysaccharides.

molecular biology↗

bioRxiv2026

Integrated serum proteomics and autoantibody analyses reveal a biomarker signature predictive of flare during biologic tapering in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by a heterogeneous clinical course with periods of remission and flare. Although biologic DMARDs (bDMARDs) have revolutionized RA treatment by enabling sustained disease control, their long-term use is associated with adverse effects and high costs, making dose tapering an attractive but clinically challenging strategy. The lack of reliable biomarkers to predict flare risk limits safe implementation of treatment de-escalation. This study aimed to identify novel circulating protein biomarkers associated with flare risk in RA patients undergoing bDMARDs tapering, useful to enable biomarker-guided treatment optimization strategies. MethodsA discovery proteomic analysis using mass spectrometry was performed on baseline serum samples from a subset of the OPTIBIO clinical trial (n=44), followed by validation in the full cohort (n=194) using ELISA. Functional pathway analysis explored biological processes associated with candidate biomarkers. In parallel, anti-cytokine autoantibodies were profiled using multiplex immunoassays. Logistic and Cox regression models were used to assess associations with flare risk. Predictive models integrating biomarkers and clinical variables were evaluated using receiver operating characteristic (ROC) analysis, sensitivity and specificity metrics, and decision curve analysis to assess clinical utility. ResultsMass spectrometry identified 806 proteins, of which 87 were differentially expressed at baseline between patients who flared and those who maintained remission during follow-up within the intervention (tapering) arm. Functional enrichment analysis highlighted immune-regulatory and innate immune pathways. Among the candidates, V-set immunoglobulin-domain-containing 4 (VSIG4) was validated as a biomarker associated with increased flare risk. Anti-interferon-{gamma} (anti-IFN{gamma}) autoantibodies were also associated with flare. A combined model including VSIG4, anti-IFN{gamma}, and the clinical variable DAS28-CRP improved predictive performance compared with clinical variables alone (AUC 0.76 vs 0.66), achieving significantly higher sensitivity. Decision curve analysis demonstrated higher net benefit of the combined model, indicating improved clinical decision-making. In a secondary analysis focused on patients with prolonged remission, representing the most suitable candidates for safe treatment tapering, the model performance further improved (AUC 0.84). ConclusionIntegration of novel serum proteomic and autoantibody biomarkers with clinical parameters improves prediction of flare during biologic tapering in RA and provides clinically relevant benefit for patient stratification. These findings support further development of biomarker-driven approaches for personalized treatment optimization strategies.

molecular biology↗

bioRxiv2022

Simulated docking reveals putative channels for the transport of long-chain fatty acids in Vibrio cholerae

Fatty acids (FA) play an important role in biological functions, such as membrane homeostasis, metabolism, and as signaling molecules. FadL is the only known protein that uptakes long-chain fatty acids in Gram-negative bacteria, and this uptake has traditionally been thought to be limited to fatty acids up to 18 carbon atoms in length. Recently however, it was found Vibrio cholerae has the ability to uptake fatty acids greater than 18 carbon atoms and this uptake corresponds to higher bacterial survivability. Using E. colis FadL as a template, V. cholerae FadL homologs vc1042, vc1043, and vca0862 have been folded, simulated on an atomistic level using molecular dynamics, and analyzed revealing the FadL transport channels. For vc1042 and vc1043 these transport channels have more structural accommodations for the many rigid unsaturated bonds of long-chain polyunsaturated fatty acids, while vca0862 was found to lack transport channels within the signature beta barrel of FadL proteins. IMPORTANCEFatty acids are important precursors for membrane phospholipids as well as energy sources for bacteria. V. choleraes uptake of long-chain fatty acids has not been studied to the atomistic level at this point, and by doing so, the resulting putative pathways show important structural details of the transport protein. Not only do the transport channels have unique differences between one another, the predicted transport mechanics of V. choleraes protein homologs differ substantially from the E. coli FadLs pathway found from X-ray crystallography studies.

molecular biology↗

bioRxiv2020

Characterization of gustatory receptor 7 in the brown planthopper reveals functional versatility

Insect pests consume tastants as their necessary energy and nutrient sources. Gustatory receptors play important roles in insect life and can form within an extremely complicated regulatory network. However, there are still many gustatory genes that have a significant impact on insect physiology, but their functional mechanism is still unknown. Here, we purified and characterized a gustatory receptor (protein) coding gene, NlGr7, from the brown planthopper (BPH) Nilaparvata lugens, which is an important insect pest of rice. Our results revealed that NlGr7 has an active association with various ligands, such as lectins, lipids (phospho- and sphingolipid) and copper. The mass-spectrometry result showed that NlGr7 is a sugar receptor, and NlGr7 is validated by different types of insoluble polysaccharides and a varied range of tastants. Furthermore, we observed that NlGr7-bound ATP hydrolysed on the ATPase activity assay, which indicated that NlGr7 may be associated with important biological functions in the BPH. The important NlGr7 for chemoreception has now been characterized in the BPH. We showed that NlGr7 in the BPH is required for various protein-ligands, as well as protein-sugars interactions, to play crucial roles in this pest. This study will provide valuable information for further functional studies of chemoreception mechanisms in this important agricultural pest.

molecular biology↗

bioRxiv2024

Reanalysis of Orbitrap Astral DIA data demonstrates the capabilities of MS/MS-free proteomics to reveal new biological insights in disease-related samples

Data-independent acquisition (DIA) became a method of choice for quantitative proteomics. With the advent of the combination of Orbitrap FTMS and asymmetric track lossless analyzer Astral these DIA capabilities were further extended with the recent demonstration of quantitative proteomic sample analyses at the speed of up to hundreds of samples per day. In particular, the dataset containing brain samples related to the multiple system atrophy was acquired using 7 and 28 min chromatography gradients and the Orbitrap Astral mass spectrometer (Guzman et al., Nat. Biotech.2024). In this work, we reanalysed the Orbitrap Astral DIA data using MS1 spectra without applying to fragmentation information using the recently introduced DirectMS1 approach. The results were compared with previous study of the same sample cohort by traditional long gradient DDA analysis. While the quantitation efficiency of DirectMS1 was comparable, we found additional five proteins of biological significance relevant to the analyzed tissue samples. Among the findings, DirectMS1 was able to detect decreased caspase activity for Vimentin protein in the multiple system atrophy samples which was barely observed from MS/MS-based methods. Our study suggests that DirectMS1 can be an efficient MS1-only addition to the analysis of DIA data in quantitative proteomic studies.

molecular biology↗

bioRxiv2025

Whole-genome methylation profiling of menstrual stem cells identifies novel biomarkers for endometriosis

Endometriosis, despite its high prevalence, is underdiagnosed and poorly managed due to lack of clinically validated biomarkers and pathophysiological insight. Menstrual blood-derived stem cells (MenSCs) have been implicated in disease pathogenesis, but their diagnostic potential remains unexplored. We conducted a clinical study (n=42; 19 endometriosis, 23 controls) to assess whether DNA methylation profiles of freshly isolated MenSCs can identify disease-specific biomarkers. Whole-genome methylation sequencing revealed differentially methylated regions (DMRs) enriched in genes linked to hallmarks of endometriosis (e.g., inflammation, tissue remodelling, development). These DMRs robustly distinguished cases from controls, independent of technical and clinical variables. Machine learning models trained and validated on these DMRs achieved high diagnostic performance (specificity 83%, sensitivity 79%). Integration with an independent single-cell RNA sequencing dataset showed that the DMRs may modulate gene expression, further supporting their biological relevance. These findings position MenSC DNA methylation profiling as a promising, non-invasive approach for early endometriosis diagnosis and personalised care.

molecular biology↗

bioRxiv2025

Rethinking Alzheimers: Novel miRNAs Illuminate a Disease Beyond the Brain

Alzheimers disease (AD) poses major health, social and economic challenges to the modern world. Despite the advances in understanding AD, our knowledge about its pathogenesis remains incomplete. Recent data suggest that circulating microRNAs (miRNAs) undergo complex changes in AD. Since these changes are yet to be comprehensively characterized, we investigated miRNAs in the context of AD using two meta-analytical approaches. We reproducibly identified 2895 miRNAs in a cohort of 4186 individuals from 22 studies. Here we show that 194 miRNAs exhibited widespread changes in AD, including some novel miRNAs not yet linked to AD. These novel AD miRNAs broaden the landscape of research on the role of miRNAs in AD. Targets of these miRNAs further uncovered many biological pathways that, to date, remain poorly understood in AD with several "AD miRNAs" never described in the brain. "AD miRNAs" described outside the brain significantly influenced interleukin signaling, Toll receptor signaling, p38 MAPK pathway and insulin/IGF pathway. Our results reveal a greater complexity of biological pathways involved in AD than previously thought and raise the question of whether AD is indeed a brain-specific and not a systemic disorder. These findings advance current understanding of AD pathogenesis and lay the ground for the development of next-generation AD biomarkers and design of miRNA-engaged therapies.

molecular biology↗

bioRxiv2026

Dynamic engagement of dual-role regulators by the Sin3 complex

Gene expression is governed by dynamic switches between repressive and activating transcriptional states1,2. Among the molecules mediating these transitions, chromatin readers and transcription factors play pivotal roles3,4. However, how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown. Here, we use an integrative structural dynamics approach - combining cryo-EM, crosslinking mass spectrometry, fragment-resolved protein interactome mapping and crystallography - to show how the dual-role chromatin reader Cti6 and transcription factors Ash1 and Ume6 engage the Sin3 deacetylase complex, a major regulatory hub in eukaryotes5. We find that Cti6 competes with Ash1 to drive its dynamic recruitment to a shared peripheral module, while Ume6 engages the Sin3 scaffold through a defined, minimal interface. Using high-throughput mutational scanning, we reveal deleterious and gain-of-function mutations in Sin3, identifying evolutionarily conserved residues essential for anchoring transcription factors. Together, these results provide structural and functional insights into how dual-role regulators engage the central Sin3 complex, revealing subtle assembly principles that may facilitate crosstalk between gene repression and activation. They also establish an integrative multidisciplinary framework to dissect the dynamics of macromolecular assemblies across biological systems.

molecular biology↗

bioRxiv2026

Transcriptomic-guided compound prioritization and proteomics validation for HNRNPU deficiency identify signalling correction

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

molecular biology↗

bioRxiv2025

Single-nucleus RNA sequencing revealed the impact of post-mortem interval on the cellular component and gene expression analysis of mouse brains

Accurate analysis of cell atlas and gene expression in biological tissues using single-nucleus RNA sequencing (snRNA-seq) is dependent on the quality of source material, and post-mortem interval (PMI) is one of the major sources of variation in RNA quality. Although the use of RNA-degraded tissues in transcriptome analysis remains controversial, such samples are sometimes the sole means to address specific questions. Current studies on the impact of PMI on transcriptome data are limited to large-scale RNA-seq, which ignores cellular heterogeneity. Thus, deciphering the non-cell- autonomous effects caused by PMI is imperative for understanding the cellular and molecular disruption it elicits. Here, we investigated the impact of PMI on cellular components and gene expression using snRNA-seq data from mouse brain tissues of post-mortem. We collected samples that were allowed to decay for varying amounts of time at 25{degrees}C prior to snRNA-seq, covering the entire range of RIN values. The different effects on the PMI to the degradation rate of mRNA and rRNA within nuclei, and the mRNA presented a more stable state. Multi-channel analysis revealed the preferential transient depletion oligodendrocytes and OPCs with increasing PMI. In addition, a rapid widespread overregulation of ribosomal transient recruitment to protein (RP) genes in various cells, and reached a plateau at PMI of 36h. Although state depletion of neuronal cells was not detected, we reported significant upregulation of PMI-dependent RP genes in its subpopulations and their cell loss. Moreover, RP genes showed the greatest differential expression in the subpopulations with greater cell perturbation, and we speculated that aberrant expression of these genes might be associated with cell death. In this study, we systematically investigated the changes in the transcriptome profile of brain tissue induced by PMI at single-cell resolution, and revealed one of the important factors that might be responsible for the changes. In addition, our data complemented a possible explanation for the changes in the cellular state of brain tissue induced by postmortem hypoxia-ischemia, and provided a reference for transcriptome studies of RNA degradation samples.

molecular biology↗

bioRxiv2021

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

molecular biology↗

bioRxiv2021

Scalable Adaptive Protein Ensemble Refinement Integrating Flexible Fitting

Recent advances in cryo-electron microscopy (cryo-EM) have enabled modeling macromolecular complexes that are essential components of the cellular machinery. The density maps derived from cryo-EM experiments are often integrated with manual, knowledge or artificial intelligence driven, and physics-guided computational methods to build, fit, and refine molecular structures. Going beyond a single stationary-structure determination scheme, it is becoming more common to interpret the experimental data with an ensemble of models, which contributes to an average observation. Hence, there is a need to decide on the quality of an ensemble of protein structures on-the-fly, while refining them against the density maps. We introduce such an adaptive decision making scheme during the molecular dynamics flexible fitting (MDFF) of biomolecules. Using RADICAL-Cybertools, and the new RADICAL augmented MDFF implementation (R-MDFF) is examined in high-performance computing environments for refinement of two protein systems, Adenylate Kinase and Carbon Monoxide Dehy-drogenase. For the test cases, use of multiple replicas in flexible fitting with adaptive decision making in R-MDFF improves the overall correlation to the density by 40% relative to the refinements of the brute-force MDFF. The improvements are particularly significant at high, 2 - 3 [A] map resolutions. More importantly, the ensemble model captures key features of biologically relevant molecular dynamics that is inaccessible to a single-model interpretation. Finally, the pipeline is applicable to systems of growing sizes, which is demonstrated using ensemble refinement of capsid proteins from Chimpanzee adenovirus. The overhead for decision making remaining low and robust to computing environments. The software is publicly available on GitHub and includes a short user guide to install the R-MDFF on different computing environments, from local Linux based workstations to High Performance Computing (HPC) environments.

biophysics↗

bioRxiv2022

Landscape configurations determining the genetic structure of the Yellow-Spotted Amazon River Turtle (Podocnemis unifilis) in Brazilian Amazonia

ContextWaterfalls and rapids of Amazon basin have been suggested as causing the speciation and genetic structure of many freshwater species, including turtles. The species behavior affects the way waterfalls and rapids limit gene flow. The Yellow-spotted River Turtle (Podocnemis unifilis), a widely distributed and endangered Amazonian turtle, does not show the habit to migrate long distances for breeding or eating, but has a complex geographic pattern of genetic variation. ObjectivesHere, we investigate isolation by distance and by resistance of P. unifilis. We analyzed if the species ecological niche and waterfalls explain the genetic distance in Brazilian Amazonia. MethodsWe evaluated the P. unifilis spatial distribution of genetic variability and diversity using the control region of mitochondrial DNA. We tested the hypotheses of isolation either by distance and resistance through an integrative approach using genetic, geographic, and ecological data. We created a resistance matrix using species niche modeling. We compared the explanation power of geographical distance (both linear and in-water distance) and resistance distance on genetic distance ({Phi}ST fixation index) using multiple regressions and Mantel tests. ResultsWe found a high genetic diversity and pattern of genetic structure proved to be geographically complex. The population structure followed some watersheds but also showed structuring within different rivers. We found that landscape resistance better explains genetic distance than linear and in-water distance. ConclusionsThe resistance of the landscape influences the displacement of individuals by aquatic, vegetational, biological, and geomorphological variables, and efforts to species conservation need to be applied throughout its distribution considering landscape genetics.

molecular biology↗

bioRxiv2022

Dietary fibre controls blood pressure and cardiovascular risk by lowering large intestinal pH and activating the proton-sensing receptor GPR65

High blood pressure (BP) is the most common cause of death globally, due to increasing the risk of cardiovascular diseases. Dietary fiber regulates BP through gut microbial production of acidic metabolites known as short-chain fatty acids (SCFAs). The specific mechanisms of how SCFAs regulate BP are still emerging. In a phenome-wide association study, we identified that the proton-sensing G-protein-coupled receptor GPR65 gene is associated with hypertension and its associated end-organ damage phenotypes. We hypothesized that acidic metabolites produced from the gut microbiota may activate GPR65, thus conferring BP regulating effects. We found that dietary fiber levels determined the luminal and interstitial tissue pH in the large intestine through production of SCFAs by the gut microbiota. We identified that low pH produced by high fiber intake, acting via GPR65 signaling, increased cAMP production and phosphorylation of CREB, and restricted the production of hypertension-promoting inflammatory cytokines by CD8+ T cells. Gpr65-/- mice spontaneously developed higher BP, cardiac and renal hypertrophy and fibrosis. We showed that the benefits of a diet high in fiber, which prevented hypertension and associated end-organ damage, were decreased in Gpr65-/- mice. Finally, adoptive transfers revealed that GPR65 deficiency in CD8+ T cells causally explained this phenotype. In conclusion, we showed that pH sensing by GPR65 in CD8+ T cells mediates much of the cardiovascular benefits of dietary fiber. pH sensing represents a novel gene-by-environment interaction of gut microbiota-to-host biological effects and may form the basis for new therapeutic strategies for hypertension.

molecular biology↗

bioRxiv2023

Comparative Genomics and Directed Evolution Reveal Genetic Determinants of Extreme UVC Radiation Tolerance in Bacteria Recovered from the Stratosphere

Aerosolized microbes surviving transport to and in the stratosphere endure extremes of low temperature, atmospheric pressure, and relative humidity, and high shortwave ultraviolet radiation flux. However, the genetic determinants for traits enabling resistance to the combination of stresses experienced by microbes in the high atmosphere have not been systematically investigated. In this study, we examined Proteobacteria and Actinobacteria isolated from the stratosphere (18 to 29 km ASL) and that demonstrated high tolerance to desiccation (15-25% RH) and UVC radiation (UVCR; {lambda}= 254 nm). Closely related reference strains were more sensitive to UVCR than the stratospheric isolates, indicating that extreme resistance is not universally distributed in these phylogenetically related bacteria. Comparative genomic analyses revealed DNA repair and antioxidant defense genes in the isolates that are not possessed by the related reference strains, including genes encoding photolyase, DNA nucleases and helicases, and catalases. Directed evolution by repeated exposure to increasing doses of UVCR improved the LD90 in a sensitive reference strain by [~]3.5-fold. The mutations acquired in Curtobacterium flaccumfaciens pv. flaccumfaciens strain DSM 20129 incrementally increased its UVCR resistance, with the accumulation of 20 point mutations in protein coding genes increasing tolerance to a level approaching that of stratospheric isolate Curtobacterium sp. L6-1. The genetic basis for the increased UVCR tolerance phenotypes observed is discussed, with a specific emphasis on the role of genes involved in DNA repair and detoxification of reactive oxygen species. ImportanceUltraviolet radiation is omnipresent in sunlight and has important biological effects on organisms. The stratosphere is the only location on Earth where microbes receive natural exposure to highly mutagenic wavelengths (<280 nm) of ultraviolet radiation. Genetic studies of bacteria from an environment that selects for extreme ultraviolet radiation resistant phenotypes has expanded what is known from studies of model species (e.g., E. coli) and identified potentially novel protection and repair strategies. In addition to deepening understanding of ultraviolet radiation photobiology in atmospheric microbes and bacteria in general, these advancements are also highly relevant to astrobiology and space biology. The cold, dry, hypobaric, and high radiation environment of the stratosphere provides an earthly analog for thin extraterrestrial atmospheres (e.g., Mars) and is ideal for bioprospecting extremophile phenotypes that enable engineering of genetic stability and functionality in bio-based space life-support systems or any application where long-term persistence is desirable (e.g., biocontrol).

molecular biology↗