Biology subjects

Prabhu, A.

Publications and source records attributed to Prabhu, A..

7 recordsLinked to original sources

bioRxiv2026

Reversible fixation of microbial cells for targeted genomics

Phylogeny- or activity-based fluorescent labelling of microorganisms coupled with fluorescence activated cell sorting (FACS) is a powerful approach to rapidly isolate cells of interest from complex communities for targeted genomics. However, the method has not been widely adopted because standard paraformaldehyde or ethanol fixation procedures hamper nucleic acid recovery and/or long-term sample storage. Here, we present dithiobis(succinimidyl propionate) (DSP) as a reversible fixative for microbial cells that enables fluorescence imaging and DNA extraction and amplification for sequencing. DSP fixation is compatible with widely used fluorescence in situ hybridization (FISH) approaches and bioorthogonal non-canonical amino acid tagging, and can be applied to diverse sample types, where it consistently performed as well as paraformaldehyde. Furthermore, high-quality genomes were obtained from individual and multiple bacterial cells selectively extracted from complex samples using FISH-FACS. Thus, DSP is a dual-use fixative for microbiome imaging and sequencing, improving targeted microbial genomics.

microbiology↗

bioRxiv2026

Molecular characterization of the heterozygous loss of function mutations in the X-linked PCDH19 gene causing PCDH19-Cluster Epilepsy

PCDH19-Cluster Epilepsy (PCDH19-CE) is a rare neurological disorder caused by mutations in the PCDH19 (Protocadherin-19) gene and is characterized by early-onset seizures and cognitive impairment. In contrast to most X-linked disorders, PCDH19 mutations predominantly affect heterozygous females, while hemizygous males are largely spared. Although advances have been made to understand the pathological mechanism underlying PCDH19-CE, key downstream targets and compensatory pathways are yet to be elucidated. Using CRISPR/Cas9 technology, we generated both a mouse model of PCDH19-CE and a human embryonic stem cell (ESC) model. Transcriptomic analysis identified genes that were differentially expressed in the brains of heterozygous (Pcdh19WT/mut) female mice compared with wildtype (WT) and homozygous (Pcdh19mut/mut) female mice. Pathway analysis of these differentially expressed genes (DEGs) revealed enrichment in pathways involved in neuronal development, ion channel activity, synaptic development and neuronal signalling. Neurons differentiated from human ESCs carrying a PCDH19 mutation exhibited similar gene expression patterns, with heterozygous neurons displaying a distinct expression pattern compared to both WT and homozygous mutant neurons. In contrast to the molecular phenotype, neurons derived from homozygous mutant cells showed highly elongated neurites while neurons from heterozygous cells showed intermediate neurite elongation. This suggests that neurite morphology correlates directly with levels of WT PCDH19. Overall, our findings indicate that heterozygous PCDH19 mutations are associated with defects in the expression of genes involved in developmental, signalling, and neuronal pathways in both mouse and human disease models, while certain morphological phenotypes appear to depend on the levels of WT PCDH19.

genetics↗

bioRxiv2026

Generation and characterization of human iPSC-derived NPC1I1061T/I10161T i3Neurons as a model for NPC1 disease

Niemann-Pick disease, type C is an autosomal recessive, fatal, neurodegenerative disorder caused by pathological variants in NPC1 or NPC2. Dysfunction of either NPC1 or NPC2 results in impaired intracellular cholesterol transport and subsequent storage of unesterified cholesterol in endolysosomal compartments. Earlier cell-based studies utilized patient fibroblasts to study this disease; however, neuronal cells allow for investigation of the neurodegenerative aspect of NPC1. Expression of neurogenin in induced pluripotent stem cells leads to the generation of i3Neurons (integrated, isogenic, and inducible), allowing for rapid, synchronized growth of homogenous neurons. In this study, we report the development and characterization of a human iPSC-derived NPC1I1061T/I1061Ti3Neuronal model system. NPC1I1061Tis a missense variant resulting in a misfolded protein targeted for proteasomal degradation in the ER. NPC1I1061T/I1061T i3Neurons phenocopied the cellular pathological features of NPC1 disease including endolysosomal cholesterol accumulation, lysosomal morphological changes, and response to the proteostasis modulator, mo56HC. The NPC1 phenotype was alleviated by 2-hydroxypropyl-{beta}-cyclodextrin treatment, a drug demonstrating efficacy both in vitro and in vivo. This NPC1I1061T/I1061T i3Neuronal cell line can facilitate future high-throughput drug and genomic screens, particularly those aimed at identifying proteostasis regulators that improve the expression/stability of the mutant NPC1 protein.

neuroscience↗

bioRxiv2026

Lets talk about sex: Male and female mice show similar fear memory retention despite hippocampal activity differences during encoding and consolidation

Accurate and efficient memory processing is essential for survival. A body of ongoing work in both human subjects and animal models suggests that memory processing may differ substantially between males and females. In mice, contextual fear memory (CFM) encoding, consolidation, and recall have been well studied, and the mouse hippocampus and amygdala have been implicated in these processes. The present study addresses whether the activation of these brain regions differs between male and female mice at each stage of CFM processing. We find that male and female C57BL6 mice show no differences in sleep behavior following single-trial contextual fear conditioning (CFC), which is essential for CFM consolidation. We also find no significant differences in CFM recall behavior between male and female mice. However, females - but not males - show significantly increased expression of cFos in dorsal hippocampal CA1 and CA2 neurons during CFM encoding. On the other hand, males - but not females - show increased cFos expression among dentate gyrus (DG) granule cells, and decreased cFos expression within dorsal CA1 and CA3 during CFM consolidation. Males also show significant decreases in dorsal CA1 cFos expression during memory recall. These findings highlight the idea that the neurobiological underpinnings of memory processing may differ between males and females, even when recall performance is identical. HighlightsHistorically, research on the neurobiological basis of memory processing has been carried out mainly in male subjects. Thus, our understanding of these mechanisms is biased towards male brain neurophysiology. This is problematic, as numerous studies have reported sex-based performance differences for episodic memory tasks, in which male subjects have variously performed better, worse, or the same as females. Here, we find that male and female mice perform similarly on a well-studied experimental memory task but nonetheless have differences in the relative activity of different brain structures during sequential stages of memory processing. This emphasizes the importance of including both males and females in memory studies, due to potential sex differences in the neurobiological substrates of memory.

neuroscience↗

bioRxiv2024

Endothelial cannabinoid CB1 receptor deficiency reduces arterial inflammation and lipid uptake in response to atheroprone shear stress

BackgroundPeripherally restricted cannabinoid CB1 receptor antagonists without central side effects hold promise for treating metabolic disorders including diabetes and obesity. In atherosclerosis, the specific effects of peripheral CB1 signaling in vascular endothelial cells (ECs) remain incompletely understood. Methods and resultsEndothelial expression of the CB1 encoding gene CNR1 was detectable in human plaque single-cell RNA sequencing data. In situ hybridization of Cnr1 in murine aortas revealed a significantly increased endothelial expression within atheroprone compared to atheroresistant regions. In vitro, CNR1 was upregulated by oscillatory shear stress (OSS) in human aortic endothelial cells (HAoECs). Endothelial CB1 deficiency (Cnr1EC-KO) in female mice on atherogenic background resulted in pronounced endothelial phenotypic changes, with reduced vascular inflammation and permeability. This resulted in attenuated plaque development with reduced lipid content in female mice, while reduced white and brown adipose tissue mass and liver steatosis were observed in both males and females. Ex vivo imaging of carotid arteries via two-photon microscopy revealed less labeled low density lipoprotein (Dil-LDL) uptake in Cnr1EC-KO. This was accompanied by a significant reduction of aortic endothelial caveolin-1 (CAV1) expression, a key structural protein involved in lipid transcytosis, in female Cnr1EC-KO mice. In vitro, pharmacological blocking with CB1 antagonist AM281 reproduced the inhibition of CAV1 expression and Dil-LDL uptake in response to atheroprone OSS in HAoECs, which was dependent on cAMP-mediated PKA activation. Conversely, the CB1 agonist ACEA increased Dil-LDL uptake and CAV1 expression in HAoECs. Finally, treatment of atherosclerotic mice with the peripheral CB1 antagonist JD-5037 reduced plaque progression, CAV1 and endothelial adhesion molecule expression in female mice. ConclusionsThese results confirm an essential role of endothelial CB1 to the pathogenesis of atherosclerosis. Peripheral CB1 antagonists may hold promise as an effective therapeutic strategy for treating atherosclerosis and related metabolic disorders. NOVELTY AND SIGNIFICANCEWhat is known? O_LIEnhanced endocannabinoid-cannabinoid CB1 receptor signaling has been implicated in metabolic disorders, atherosclerosis, and hypertension, but the cell-specific role of endothelial CB1 in atherosclerosis is not well understood. C_LIO_LIGlobal CB1 antagonists improve metabolic function and inhibit atherosclerotic plaque development in mouse models, but have failed in the clinic due to centrally mediated psychiatric side effects. C_LI What is new? O_LIBased on human single-cell RNA sequencing data, CB1 is expressed in human plaque ECs. C_LIO_LIUsing transgenic mouse models and human primary aortic endothelial cells, we provide evidence for a key role of CB1 in endothelial shear stress response, inflammatory gene expression, and LDL uptake. C_LIO_LIThe underlying signaling pathway of CB1-induced endothelial LDL uptake involves a cAMP-PKA-dependent regulation of caveolin 1 (CAV1) expression, a structural protein of the shear stress sensitive signaling domains of the plasma membrane. C_LIO_LIBy limiting endothelial CAV1 and VCAM1 expression, peripherally restricted CB1 antagonists confer atheroprotection in mice. C_LI Our findings reveal that endothelial CB1 expression is induced by atheroprone shear stress responses and contributes to impaired vascular barrier function, inflammation, and lipid uptake, thereby promoting atherosclerotic lesion formation and progression. By elucidating the transcriptomic pathways regulated by endothelial CB1 and its broad influence on lipid uptake and metabolism in arteries, liver, and brown and white adipose tissue, our study provides insights into novel pathways and potential interventions for the treatment of atherosclerosis and metabolic disorders. The use of peripherally restricted CB1 antagonists that specifically target vascular inflammation and tissue lipid storage could be a complementary and safe therapeutic avenue to treat cardiovascular and metabolic disease comorbidities without altering CB1 signaling in the brain.

immunology↗

bioRxiv2022

CorA gene rearrangement triggered the salinity-driven speciation of Poseidoniales

Land-ocean interactions greatly impacted the evolution of coastal life on Earth. However, the geological forces and genetic mechanisms that shaped evolutionary adaptations and allowed microorganisms to inhabit coastal brackish waters remain largely unexplored. Here, we infer the evolutionary trajectory of the ubiquitous heterotrophic archaea Poseidoniales (Marine Group II archaea) across global aquatic habitats. Our results show that their brackish subgroups have evolved through the rearrangement of the magnesium transport gene corA that conferred osmotic-stress tolerance dated to over 600 million years ago. The coastal family of Poseidoniales showed a rapid increase in the evolutionary rate during and in the aftermath of the Cryogenian Snowball Earth (~700 million years ago), possibly in response to the enhanced phosphorus supply and the rise of algae. Our study highlights the close interplay between genetic changes and ecosystem transformations that boosted microbial diversification in the Neoproterozoic continental margins.

evolutionary biology↗

bioRxiv2022

Insights into plastic biodegradation: community composition and functional capabilities of the superworm (Zophobas morio) microbiome in styrofoam feeding trials

Plastics are inexpensive and widely used organic polymers, but their high durability hinders biodegradation. Polystyrene, including extruded polystyrene also known as styrofoam, is among the most commonly produced plastics worldwide and is recalcitrant to microbial degradation. In this study, we assessed changes in the gut microbiome of superworms (Zophobas morio) reared on bran, polystyrene, or under starvation conditions over a three weeks time period. Superworms on all diets were able to complete their life cycle to pupae and imago, although superworms reared on polystyrene had minimal weight gains, resulting in lower pupation rates. The change in microbial gut communities from baseline differed considerably between diet groups, with polystyrene and starvation groups characterized by a loss of microbial diversity and the presence of opportunistic pathogens. Inferred microbial functions enriched in the polystyrene group included transposon movements, membrane restructuring, and adaptations to oxidative stress. We detected several encoded enzymes with reported polystyrene and styrene degradation abilities, supporting previous reports of polystyrene degrading bacteria in the superworm gut. By recovering metagenome-assembled genomes (MAGs) we linked phylogeny and functions and identified genera including Pseudomonas, Rhodococcus and Corynebacterium, that possess genes associated with polystyrene degradation. In conclusion, our results provide the first metagenomic insights into the metabolic pathways used by the gut microbiome of superworms to degrade polystyrene. Our results also confirm that superworms can survive on polystyrene feed, however, this diet has considerable negative impacts on host gut microbiome diversity and health. Impact StatementIncreasing plastic pollution is a major environmental problem, and a recently proposed way to counteract this trend is to embrace a circular economy, in which used materials are recycled, rather than disposed of. An important step to facilitate this process is to invent new approaches for upcycling of plastic waste to desirable consumer products. Microbial plastic degradation and conversion is likely to play a considerable part in shaping a circular economy, by engineering microbes or their enzymes to bio-upcycle plastic waste. A first step towards actualizing this goal is to identify microbes that can degrade polystyrene and to investigate the enzymes and pathways involved. Our study represents the first metagenomic analysis of an insect gut microbiome on a polystyrene diet. It identifies bacteria with polystyrene and styrene degrading abilities, and infers enzymes and pathways involved in these reactions. Therefore, our results contribute towards understanding microbial polystyrene degradation and will provide a base for future investigations into microbial upcycling of plastic waste.

microbiology↗