Biology subjects

John, J.

Publications and source records attributed to John, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Development and validation of a genome-informed multiplex PCR for specific detection of typhoidal Salmonella serovars

Enteric fever caused by Salmonella enterica serovars Typhi and Paratyphi A, B and C remains a major public health burden in endemic regions. Existing molecular assays frequently demonstrate limited specificity due to cross-reactivity with non-typhoidal Salmonella (NTS). In this study, we developed and validated a genomics-informed multiplex PCR assay capable of simultaneously differentiating all four typhoidal Salmonella serovars. A curated dataset of 3,239 Salmonella genomes, including S. Typhi (n=361), S. Paratyphi A (n=453), S. Paratyphi B (n=511), S. Paratyphi C (n=62), and NTS genomes (n=1,853), was used for comparative genomic analysis. Thirty published PCR targets were evaluated in silico, followed by pangenome and SNP analyses to identify discriminatory loci for mismatch amplification mutation assay (MAMA)-based primer design. Candidate primers were validated using in silico PCR, BLASTn analysis, and laboratory testing against a panel of typhoidal Salmonella, clinical NTS isolates, and non-Salmonella bacterial pathogens. In silico evaluation demonstrated substantial cross-reactivity among many published targets, whereas SNP-informed primer design targeting staG (S. Typhi), SPA0152 (S. Paratyphi A), SPAB_03490 (S. Paratyphi B), and SPC_0571 (S. Paratyphi C) achieved predicted specificities of 98-100% while retaining high analytical sensitivity (>97%) across target genomes. Combined with a pan-Salmonella invA target, the multiplex assay precisely identified all target serovars in vitro with minimal cross-reactivity. These findings demonstrate that genomics-informed SNP-based primer design enables reliable multiplex differentiation of typhoidal Salmonella serovars and provides a scalable framework for improving enteric fever diagnosis and surveillance in endemic settings. ImportanceTyphoidal Salmonella serovars remain major causes of enteric fever in endemic regions, yet molecular differentiation from non-typhoidal Salmonella (NTS) remains challenging because of extensive genomic conservation and cross-reactivity of commonly used diagnostic targets. In this study, we combined large-scale comparative genomics of 3,239 Salmonella genomes with SNP-informed primer design to develop a multiplex PCR assay capable of simultaneously differentiating all four typhoidal serovars (S. Typhi, S. Paratyphi A, B, and C) from NTS and other non-Salmonella pathogens. Unlike conventional gene-content-based assays, this approach incorporated lineage-specific SNPs and mismatch amplification strategies to improve specificity while maintaining high analytical sensitivity. In silico evaluation demonstrated high diagnostic performance across diverse global lineages, while in vitro testing confirmed accurate serovar-level discrimination with minimal cross-reactivity. These findings demonstrate the value of population-scale genomics for molecular assay development and provide a scalable framework for improving diagnosis and surveillance of enteric fever in endemic settings.

molecular biology↗

bioRxiv2026

Environmental Gradients Shape the Hydrocarbon-Degrading Microbiome in Two Mid Atlantic Bays.

Hydrocarbons are recalcitrant organic matter that are released into the environment via natural and anthropogenic activities. We hypothesized that abiotic and biotic factors, including salinity, temperature, seasonality, microbial interactions, and functional redundancy, influence the abundance and activity of potential hydrocarbon degraders in the Delaware and Chesapeake Bays. We identified key genes in hydrocarbon degradation pathways in metagenomes, metatranscriptomes, and metagenome assembled genomes (MAGs) from these estuaries. Aerobic aromatic and alkane degradation pathways predominated in both estuaries, with higher gene abundances observed in low-salinity spring and summer samples. Hydrocarbon degrading MAG abundance were significantly structured by salinity, temperature, nitrate, and silicate concentrations. Metatranscriptomic analyses revealed consistently higher expression of aerobic alkane and aromatic degradation genes in the Delaware compared to the Chesapeake Bay, with the highest occurring under low-salinity spring conditions in the former. Catechol degradation pathways exhibited high functional redundancy, whereas the naphthalene degradation pathway showed restricted distribution. Co-expression analysis revealed that Burkholderiales displayed condition dependent metabolic coupling while Pseudomonadales integrated hydrocarbon degradation with fermentation and central metabolism, demonstrating complementary strategies that support multi-scale ecosystem resilience. In conclusion, environmental gradients and taxon-specific metabolic strategies together govern hydrocarbon degradation potential in these estuaries, with implications for predicting ecosystem responses to hydrocarbon inputs under changing conditions. ImportanceCoastal estuaries are among the most contaminated aquatic environments on Earth, receiving continuous hydrocarbon inputs from industrial activity, urban runoff, and natural sources. Microorganisms are the primary agents of hydrocarbon breakdown in these systems yet predicting when and where this capacity is active and how resilient it is to environmental change remains a major challenge. Using paired genomic and transcriptomic data from microbial genomes across two major mid-Atlantic estuaries, we show that hydrocarbon degradation capacity is not uniformly distributed but is instead shaped by salinity, nutrients, and seasonality in pathway-specific ways. Critically, dominant degrader taxa employ fundamentally different metabolic strategies to sustain this function across fluctuating conditions, providing a form of community-level insurance against environmental disturbance. These findings advance our ability to predict microbial hydrocarbon degradation in coastal systems and inform nature-based approaches to bioremediation under increasing climate and anthropogenic pressures.

genomics↗

bioRxiv2026

Low-barrier hydrogen-bond powers long-range radical transfer in the metal-free ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 [A]) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combing multiscale quantum and classical simulations with directed mutagenesis, x-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable DOPA initiator radical. We show that DOPA* is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis. Significance StatementRibonucleotide reductases (RNRs) are ancient enzymes responsible for the synthesis of deoxyribonucleotides from ribonucleotides. RNRs catalyze this reaction via a long-range proton-coupled electron transfer (PCET) process, involving the formation of a stable protein radical. Yet, despite decades of detailed structural, biochemical, spectroscopic and computational studies, the mechanistic principles of this process remain unclear and much debated. Here, we show that metal-free RNRs power the reduction of RNA building blocks by a highly stable organic DOPA initiator radical, arising from a unique low-barrier hydrogen bonding (LBHB) network that enables the radical transport by strong redox-tuning effects. Our findings reveal mechanistic principles underlying the elusive PCET reactions of metal-free RNRs, and provide evidence for the involvement of quantum effects in enzyme catalysis.

biophysics↗

bioRxiv2026

PRDX6 Modulates Immune Checkpoint Inhibitor Response by Antagonizing Ferroptosis Induced By HDAC Inhibitors

Therapeutic resistance limits the efficacy of histone deacetylase (HDAC) inhibitors and immune checkpoint therapies in cancer. While HDAC inhibitors can induce ferroptosis, tumor cells often evade this cell death via antioxidant defenses. Here we identify peroxiredoxin 6 (PRDX6) as a critical modulator of resistance to HDAC inhibitor largazole by suppressing ferroptosis through its phospholipase A2 activity and maintaining GPX4 expression. Using genome-wide CRISPR activation screening, biochemical assays, and syngeneic tumor models, we show that PRDX6 depletion enhances largazole-induced lipid peroxidation, ferroptotic stress, and reshapes the tumor microenvironment to promote T-cell infiltration and inflammatory cytokine release. Importantly, combining PRDX6 knockdown with HDAC inhibition potentiates anti-PD-L1 immunotherapy efficacy and prolongs survival in vivo. These findings reveal PRDX6 as a redox gatekeeper linking ferroptosis resistance to immune evasion and suggest that co-targeting PRDX6 and HDAC pathways may improve responses to cancer immunotherapy.

cancer biology↗

bioRxiv2026

FPmotion an Automated Signal Processing and Statistical Analysis Tool for Fiber Photometry Data

Fiber photometry measures neural activity in vivo from genetically encoded indicators. Recordings generate large datasets that require extensive preprocessing and robust statistical methods for meaningful interpretation. However, existing analysis tools demand programming expertise, limiting accessibility. Here we describe FPmotion, a comprehensive, user-friendly software platform for batchwise processing, integration, and statistical analysis of fiber photometry data with or without accompanying behavioral information. FPmotion performs filtering, isosbestic regression, {Delta}F/F computation, detrending, and z-scoring. It also extracts detailed peak properties at whole-file, block-level, and individual-peak resolution. When behavioral data is provided, FPmotion automatically identifies behavioral bouts, computes bout-level statistics, performs peri-event signal extraction, and supports multi-group comparisons through ANOVA- and LMEM-based statistical frameworks. All analyses generate publication-ready figures and structured CSV outputs suitable for downstream workflows. FPmotion also introduces a dedicated alignment module for peri-event signal analysis. This module applies dynamic time warping followed by barycenter averaging to realign peri-event traces while preserving the behavioral time anchor, producing cleaner and more temporally coherent peri-event motifs. We demonstrate FPmotions capabilities using the dlight1.1 sensor to measure dopamine responses from ventral striatum in behaving mice before and after amphetamine treatment. Together, FPmotion offers a fully automated framework for FP data analysis that improves interpretability and accessibility while reducing analysis time substantially.

neuroscience↗

bioRxiv2025

Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

BackgroundWhile roles for canonical autophagy in the pathophysiology of cardiovascular disease have been established, we have limited understanding of the non-canonical functions of autophagy proteins in this context. LC3-asssociated endocytosis (LANDO) is a novel non-canonical function of autophagy proteins, in which LC3 (microtubule-associated protein light chain 3) is conjugated to early endosome membranes using a portion of the canonical autophagy machinery, and functions in the endocytic recycling of several plasma membrane proteins. Here we ask whether perturbation of LANDO can promote cardiovascular pathogenesis. MethodsCardiac and endothelial functions were assessed by echocardiography and flow-mediated dilatation in mice lacking Rubicon (Rubcn-/-) or the WD domain of ATG16L1 (Atg16l1{Delta}WDki), two known effectors of LANDO. Mice with conditional depletion of Rubicon in the endothelial, myeloid and cardiomyocyte compartments were used as well. Three-dimensional murine cardiac vasculature leakiness was investigated by light sheet fluorescence microcopy. Endothelial activation induced by shear stress was characterized in vitro in primary endothelial cells isolated from murine lungs and human aortic endothelial cells. Associations between genetically predicted expression of candidate genes involved in LANDO and human cardiovascular parameters were studied in the Young Finns Study and the UK Biobank. ResultsCompared to littermate controls, young Rubcn-/- and Atg16l1{Delta}WDki mice showed a decrease in cardiac and endothelial functions, as did mice with endothelium-specific deficiency. VEGFR2 recycling to the plasma membrane and nitric oxide pathway during shear stress were disrupted in LANDO-deficient primary murine and human endothelial cells. Proteomic analysis in primary human aortic endothelial cells revealed an upregulation of intracellular hemoglobin subunit alpha (Hb-) upon shear stress, which was blunted when RUBCN was ablated. Genetic expression studies uncovered several candidate genes related to LANDO that correlated with cardiovascular parameters. These included the retromer complex subunit VPS29, disruption of which decreased Hb- expression levels in human endothelial cells. ConclusionsOur data support a pivotal role of non-canonical functions of autophagy proteins in recycling VEGFR2 upon shear stress activation in endothelial cells together with Hb- expression that may contribute to the etiology of cardiovascular diseases.

physiology↗

bioRxiv2025

Five years of national airborne pollen monitoring in South Africa: Biome-specific calendars to inform allergy diagnosis and prevention

Pollen monitoring is crucial for understanding seasonal patterns, supporting allergy diagnosis and informing early-warning tools to mitigate allergic diseases. The Southern Hemisphere lacks long-term data on pollen seasons, with extremely few from Africa. We present pollen calendars based on five-year data from the South African Pollen Monitoring Network (SAPNET). Airborne pollen from 2019 to 2024 in biomes across South Africa was collected using Hirst-type volumetric spore traps and standard protocols. Daily concentrations were analysed by light microscopy. The five-year mean annual pollen integral (APIn, pollen grains/m3 per year) was calculated for each site. The five-year mean APIn was highest in the Grassland Biome (Bloemfontein, 11654 pg/m3; range 8515 to 14454 pg/m3) and lowest in the Albany Thicket Biome (Gqeberha, 1372; range 853 to 2010 pg/m3). The Grassland Biome (Johannesburg) had the highest averaged tree pollen concentration (7558; range 6575 to 8803 pg/m3). The Savanna Biome (Kimberley) had the highest average grass pollen concentration (3150; 1826 to 3785 pg/m3). The grass family (Poaceae) was the most common pollen type across all biomes. Other common contributing taxa were exotic trees Cupressaceae, Platanus, Morus and Betula. Tree seasons were July to September, whilst grass and weed pollen seasons varied across the different biomes. These five-year pollen calendars provide the first biome-specific national reference for airborne pollen exposure in South Africa. The findings provide baseline data for the clinical management of allergic disease.

ecology↗

bioRxiv2025

Ecological distribution, environmental roles and drivers of Actinobacteriota in two Mid-Atlantic estuaries

Actinobacteriota, a bacterial phylum renowned for members that produce bioactive compounds (e.g., antibiotics), has key roles in terrestrial and aquatic ecosystems. Although soil and marine/freshwater Actinobacteriota are well studied, functions and activities of their estuarine counterparts are poorly understood. We characterized 67 metagenome-assembled genomes (MAGs) belonging to 12 Actinobacteriota families from Chesapeake and Delaware Bay water samples across different seasons, salinities, and size fractions. MAGs from four dominant families, Ilumatobacteraceae, Nanopelagicaceae, Microbacteriaceae, and S36-B12, were examined in depth for their abundance, functional potential, estimated growth rates, and gene expression among samples. Actinobacteriota were most abundant in low- to medium-salinity samples during spring and summer. Their abundance patterns were strongly influenced by combinations of salinity, temperature, and phosphate, nitrate and silicate concentrations. Notably, many exhibited high estimated growth rates under low and medium salinities in summer. Members of the four major families showed a range of metabolic capacities from generalist to specialist, and all encoded biosynthetic gene clusters (BGCs) for secondary metabolites, particularly terpenes and betalactones, that were differentially expressed across conditions. Bay, salinity and size fraction were the primary drivers of gene expression differences. Distinct secondary metabolite genes were expressed between bays, with higher expression generally observed in medium compared to low salinities. These findings underscore the metabolic versatility and environmental responsiveness of Actinobacteriota, highlighting their active role in estuarine microbial communities and their contributions to biogeochemical cycling in dynamic coastal ecosystems. ImportanceEstuaries are dynamic transition zones where microbial communities play key roles in nutrient cycling and ecosystem functioning. This study provides new insights into the distribution of Actinobacteriota families and their responses to salinity and nutrient gradients in two major U.S. estuaries, the Chesapeake and Delaware Bays. We characterized the potential roles of Actinobacteriota in biogeochemical processes and secondary metabolite production, which may influence broad microbial interactions. These bacteria may also serve as a source of novel bioactive compounds, including antibiotics. By integrating omic and environmental data, this work advances our understanding of Actinobacteriota adaptations and their ecology in estuarine environments.

microbiology↗

bioRxiv2025

Forest biogeochemical monitoring indicates altered microbial communities, macronutrient availability, CO2 emissions and litter chemistry in root zone soil of oak trees with Acute Decline symptoms

Acute and Chronic Oak Decline (AOD & COD) widely impact oak woodlands across Europe. AOD is known to cause rapid decline in tree health in a short span of 3-5 years. However, the interactions between such oak decline and soil biogeochemical dynamics are not fully understood. We selected three oak trees for each of the three treatments for biogeochemical monitoring: 1. No symptoms (Healthy), 2. AOD & 3. COD symptoms, in Writtle woodlands, Essex, UK. The selected trees were used for quarterly root zone soil sampling (cores to depth of 40 cm), and monthly surface soil (0-10 cm), leaf litter and GHG emissions sampling over a year (2022-23). Soil samples were characterised for their physico-chemical properties, total nutrient content & availability. Leaf litter samples were sorted into branches, leaves, seeds and flowers, weighed and their chemistry characterised. Soil microbial communities were characterised using phospholipid fatty acid analyses. Almost all the measured properties showed significant changes with season and depth, indicating a strong temporal and depth effect on forest soil biogeochemical dynamics. Notably macro-nutrient contents of leaf litter were greater in the summer period, which resulted in increased soil nutrient availability in autumn. This pattern was particularly exaggerated in AOD trees, which had greater nutrient content in litterfall in summer and greater nutrient availability in root zone soil in autumn than those of COD and Healthy trees. We found that AOD root zone soil have altered microbial communities, decreased CO2 emissions and increased macronutrient availability. Taken together, our study shows poor physiological nutrient management by diseased AOD trees revealed by greater nutrient concentration in AOD leaf litter indicating the lack of nutrient resorption and re-allocation before leaf senescence, and less active root zone soil under AOD trees, shown by reduced soil CO2 emissions and increased nutrient availability in oak root influenced zones. Hence, we conclude that the biogeochemical dynamics of AOD root zone soils are significantly different to that of healthy and COD trees, however controlled experiments are needed alongside these field observations to disentangle the causes from effects in understanding the relationship between soil health and oak decline. HighlightsO_LIWe monitored forest soil biogeochemical dynamics under oak trees of different health status C_LIO_LIAcute Oak Decline (AOD) trees had greater macronutrient availability in root zone soils C_LIO_LIAOD trees had greater nutrient content in litter added to the root zone soil C_LIO_LIAOD root zone soils had altered microbial community structure & reduced CO2 emissions C_LIO_LIWe conclude that AOD trees have poor physiological nutrient management and less active rhizosphere compared to healthy trees. C_LI

ecology↗

bioRxiv2025

Functional profiling of KCNE1 variants informs population carrier frequency of Jervell and Lange-Nielsen syndrome type 2

Congenital long-QT syndrome (LQTS) is most often associated with pathogenic variants in KCNQ1 encoding the pore-forming voltage-gated potassium channel subunit of the slow delayed rectifier current (IKs). Generation of IKs requires assembly of KCNQ1 with an auxiliary subunit encoded by KCNE1, which is also associated with LQTS but causality of autosomal dominant disease is disputed. By contrast, KCNE1 is an accepted cause of recessive type 2 Jervell and Lange-Nielson syndrome (JLN2). The functional consequences of most KCNE1 variants have not been determined and the population prevalence of JLN2 is unknown. Methods: We determined the functional properties of 95 KCNE1 variants co-expressed with KCNQ1 in heterologous cells using high-throughput voltage-clamp recording. Experiments were conducted with each KCNE1 variant expressed in the homozygous state and then a subset was studied in the heterozygous state. The carrier frequency of JLN2 was estimated by considering the population prevalence of dysfunctional variants. Results: There is substantial overlap between disease-associated and population KCNE1 variants. When examined in the homozygous state, 68 KCNE1 variants exhibited significant differences in at least one functional property compared to WT KCNE1, whereas 27 variants did not significantly affect function. Most dysfunctional variants exhibited loss-of-function properties. We observed no evidence of dominant-negative effects. Most variants were scored as variants of uncertain significance (VUS) and inclusion of functional data resulted in revised classifications for only 14 variants. The population carrier frequency of JLN2 was calculated as 1 in 1034. Peak current density and activation voltage-dependence but no other biophysical properties were correlated with findings from a mutational scan of KCNE1. Conclusions: Among 95 disease-associated or population KCNE1 variants, many exhibit abnormal functional properties but there was no evidence of dominant-negative behaviors. Using functional data, we inferred a population carrier frequency for recessive JLN2. This work helps clarify the pathogenicity of KCNE1 variants.

genetics↗

bioRxiv2024

JNK1 And Downstream Signalling Hubs Regulate Anxiety-Like Behaviours In A Zebrafish Larvae Phenotypic Screen

Current treatments for anxiety and depression show limited efficacy in many patients indicating that research into new underlying mechanisms is needed. Inhibition of JNK1 has been shown to evoke an anxiolytic-and antidepressant-like phenotype in mice however the downstream effectors that elicit these behavioural effects are unknown. Here we employ a zebrafish (D. Rerio) larvae behavioural assay to identify an antidepressant-/anxiolytic-like phenotype based on 2759 measured stereotypic responses to clinically proven antidepressant and anxiolytic (AA) drugs. Employing machine learning, we classify an AA phenotype from behavioural features measured during and after a startle battery in fish exposed to AA drugs (fluoxetine, imipramine, diazepam, lithium chloride, ketamine). We demonstrate that structurally independent JNK inhibitors replicate the AA classification with high accuracy, consistent with findings in mice. We go on to identify signalling hubs downstream from JNK1 by comparing phosphoproteome data from wildtype and Jnk1-/- mouse brains, and test these hubs as possible mediators of the AA phenotype in zebrafish larvae. Among these, we find that AKT, GSK-3, 14-3-3{zeta}/{varepsilon} and PKC{varepsilon}, when pharmacologically targeted, phenocopy clinically proven AA drugs. This assay shows promise as an early phase screening for compounds with anti-stress-axis/anxiolytic-like properties, and for mode of action analysis.

pharmacology and toxicology↗

bioRxiv2023

Vascularized Liver Tissue Embedded Bioprinting Utilizing GelMA/Nanoclay-based Composite hydrogels

As the aging population grows, the need to regenerate non-self-repairing tissues becomes increasingly crucial for enhancing our quality of life. Tissue engineering offers a promising solution, particularly in recreating the intricate networks of blood vessels crucial for tissue vitality. These tissues rely on effective nutrient and oxygen circulation, with an optimal oxygen diffusion range of 100-200 {micro}m. Yet, crafting vascularized in vitro tissues remains a significant challenge. This study addresses the challenge by using GelMA-based hydrogels as a photocrosslinkable support bath, a biocompatible and versatile choice for biological applications. To enhance the rheological properties for in vitro tissue engineering, Laponite (LPN) is introduced as a rheology modifier. The study optimizes the GelMA-LPN nanocomposite hydrogel composition, ensuring the desired physical, mechanical, and rheological properties, including recovery. The research also explores the biological implications, encapsulating liver cells within the nanocomposite hydrogel, and studying their behavior under perfusion conditions. This research presents a promising avenue for creating vascularized in vitro tissues, potentially advancing tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2023

Recent emergence of cephalosporin resistant Salmonella Typhi carrying IncFIB(K) plasmid encoding blaCTX-M-15 gene in India

The emergence and spread of Salmonella Typhi (S. Typhi) resistant to third-generation cephalosporins is a serious global health concern. In this study, we genomically characterized 142 cephalosporin-resistant S. Typhi strains isolated from India. Comparative genome analysis revealed the emergence of a new clone of ceftriaxone-resistant S. Typhi harboring three plasmids of the incompatibility groups IncFIB(K), IncX1, and IncFIB(pHCM2). Among these, the IncFIB(K) plasmid confers resistance to third-generation cephalosporins through the blaCTX-M-15 gene, along with other resistance determinants such as aph(3"), aph(6), sul2, dfrA14, qnrS, and tetA. Phylogenetic analysis showed that the isolates from Gujarat (n=140/142) belong to a distinct subclade (genotype 4.3.1.2.2) within genotype 4.3.1.2 (H58 lineage II). SNP-based phylogenetic analysis of the core genes in IncFIB(K) reveals a close genetic relationship between the plasmid backbone and that of IncFIB(K) from other Enterobacterales, suggesting that the H58 lineage II possesses the capability to acquire MDR plasmids from these organisms. This could indicate the potential onset of a new wave of ceftriaxone-resistant S. Typhi in India. The implementation of control measures such as vaccination, and improved water, sanitation, and hygiene (WASH) systems, is crucial in areas where MDR or XDR S. Typhi strains are prevalent to curb the spread and impact of these resistant strains. ImportanceTyphoid fever remains a significant public health problem in many parts of the world, particularly in resource-limited settings with inadequate sanitation and limited access to clean water. The emergence of MDR S. Typhi resistant to first-line antibiotics (chloramphenicol, ampicillin, and cotrimoxazole) and fluoroquinolones marked the initial treatment challenge, leading to reliance on azithromycin and third-generation cephalosporins. Recently, researchers identified the emergence of XDR typhoid in Pakistan that is resistant to five classes of antibiotics including third-generation cephalosporins. In this study, we report a recent emergence of S. Typhi isolates resistant to third-generation cephalosporins. Genomic characterization and evolutionary analysis of the new strains revealed a recent acquisition of blaCTX-M carrying IncFIB(K) plasmid led to the emergence of ceftriaxone-resistant S. Typhi. Due to the ongoing nature of this outbreak, the data from this study deserves further consideration in order to control its spread in India.

genomics↗

bioRxiv2023

Loss of secondary motor cortex neurons in chronic neuropathic pain

Chronic neuropathic pain is associated with structural plasticity of the brain on different spatial scales, yet, little is known on the mesoscopic scale of tissue composition. Here, we determined the cellular composition of cortical areas and structural variability of entire neurons during the development of chronic neuropathic pain using longitudinal in vivo two-photon microscopy and behavioral assessment. When monitoring cell type composition in response to spared-nerve injury in 84 cortical volumes containing {bsim}25000 cells each, we found neuronal loss in the secondary motor cortex region M2 immediately adjacent to the cingulate cortex already one week after surgery. Loss of mostly interneurons was also evident when monitoring individual M2 neurons over time. This neuronal loss was preceded by decreased spine density and loss of distal dendritic branches. In conclusion, our work delineates M2 as a novel site and neuronal loss as a so far underappreciated mechanism underlying chronic neuropathic pain states.

neuroscience↗

bioRxiv2022

Comprehensive monitoring of tissue composition using in vivo imaging of cell nuclei and deep learning

Comprehensive analysis of tissue composition has so far been limited to ex-vivo approaches. Here, we introduce NuCLear (Nucleus-instructed tissue composition using deep learning), an approach combining in vivo two-photon imaging of histone 2B-eGFP-labeled cell nuclei with subsequent deep learning-based identification of cell types from structural features of the respective cell nuclei. Using NuCLear, we were able to classify almost all cells per imaging volume in the secondary motor cortex of the mouse brain (0.25 mm3 containing [~]25000 cells) and to identify their position in 3D space in a non-invasive manner using only a single label throughout multiple imaging sessions. Twelve weeks after baseline, cell numbers did not change yet astrocytic nuclei significantly decreased in size. NuCLear opens a window to study changes in relative abundance and location of different cell types in the brains of individual mice over extended time periods, enabling comprehensive studies of changes in cellular composition in physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2022

De novo discovery of traits co-occurring with chronic obstructive pulmonary disease

Epidemiological research indicates that chronic obstructive pulmonary disease (COPD) is a heterogeneous group of chronic lung conditions that are typically accompanied by cardiovascular disease, depression, lung cancer and other conditions. Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) associated with COPD and the co-occuring conditions, suggesting common biological mechanisms underlying COPD and these co-occuring conditions. To identify them, we have integrated information across different biological levels (i.e. genetic variants, lung-specific 3D genome structure, gene expression and protein-protein interactions) to build lung-specific gene regulatory and protein-protein interaction networks. We have queried these networks using disease-associated SNPs for COPD, unipolar depression and coronary artery disease. Our results show that COPD-associated SNPs can control genes involved in the regulation of lung or pulmonary function, asthma, brain region volumes, cortical surface area, depressed affect, neuroticism, Parkinsons disease, white matter microstructure and smoking behaviour. We describe the regulatory connections, genes and biochemical pathways that underly these co-occuring trait-SNP-gene associations. Collectively, our findings provide new avenues for the investigation of the underlying biology and diverse clinical presentations of COPD. In so doing, we identify a collection of genetic variants and genes that may aid COPD patient stratification and treatment.

genomics↗

bioRxiv2022

Genomic analysis unveils the role of genome degradation events and gene flux in the emergence and persistence of S. Paratyphi A lineages

Paratyphoid fever caused by S. Paratyphi A is endemic in parts of Asia and Sub-Saharan Africa. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017-2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis documented that the genome degradation events and gene acquisitions or losses play a major role in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 pseudogene-forming mutations possibly associated with the emergence of lineages were identified. Pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We also identified that the six characteristic missense mutations associated with the lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A confer only a low structural impact and would therefore have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and intervention by means of genomic surveillance would be continually needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today.

genomics↗

bioRxiv2022

Redox-controlled structural reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

biochemistry↗