Biology subjects

Manivannan, S.

Publications and source records attributed to Manivannan, S..

10 recordsLinked to original sources

bioRxiv2026

PBF/PTTG1IP coordinates focal adhesion formation, polarity and cell motility

Directional cell migration requires cells to sense extracellular cues and coordinate adhesion, cytoskeletal remodelling and polarity, yet the molecular regulators that integrate these events remain incompletely defined. PTTG1-binding factor (PBF/PTTG1IP) is a transmembrane glycoprotein extensively characterised in pathological overexpression and cancer models, where elevated expression promotes tumourigenic cellular phenotypes. However, its endogenous physiological function remains poorly understood. Here, separate enrichment analyses of transcriptomic and phosphoproteomic profiles from PBF-overexpressing cells collectively highlighted cell adhesion, extracellular matrix organisation, cytoskeletal regulation and cell motility as major PBF-associated programmes. Using a novel Pbf knockout mouse model, we show that primary Pbf-KO mouse embryonic fibroblasts exhibit impaired migration and invasion. PBF loss reduced fibronectin adhesion, disrupted focal adhesion number, size and distribution, attenuated FAK Tyr397 phosphorylation and delayed early adhesion formation. Live-cell imaging and Golgi orientation assays further revealed altered actin organisation and impaired front-rear polarity in Pbf-KO cells. Key phenotypes were conserved in CRISPR-Cas9 PBF-KO human thyroid cancer cells, while targeted PBF re-expression restored migration in Pbf-KO MEFs. We thus identify PBF as an endogenous regulator of cell adhesion, polarity and directional motility, providing a framework for understanding how pathological PBF overexpression may promote invasive behaviour in cancer.

cell biology↗

bioRxiv2025

Ventral Tegmental Area Glutamatergic Neurons Suppress Hippocampal Sharp-wave Ripples and Induce Head Movements in Mice

Sharp-wave ripples are brief, high-frequency hippocampal oscillations crucial for memory consolidation, occurring predominantly during the non-rapid-eye-movement (NREM) sleep. While ripples are generated intrinsically within the hippocampus, growing evidence suggests that their occurrence is modulated by subcortical regions. Whether the ventral tegmental area (VTA)--a subcortical region that directly projects to the hippocampus--regulates ripple activity remains unknown. To investigate this, we optogenetically activated VTA neurons during NREM sleep and recorded ripple activity in the dorsal hippocampus via field recording electrodes. Using cell-type specific excitatory opsin expression in the VTA, we found that activation of glutamatergic (vGlut2+) neurons strongly suppressed ripple incidence. In contrast, activation of GABAergic (vGAT+) neurons produced weak suppression, and activation of dopaminergic (DAT+) neurons showed no effect. During ripple suppression by glutamatergic activation, we observed small but consistent head movements. Trial-by-trial analysis revealed no correlation between head movement and ripple suppression. Furthermore, glutamatergic activation during wakefulness also led to head movement, suggesting that ripple suppression and head movement could be dissociated. Overall, our results suggest that VTA vGlut2+ neurons play a role in head movement and that their activation suppresses hippocampal sharp-wave ripples during NREM sleep, offering a potential way to alter memory consolidation.

neuroscience↗

bioRxiv2024

Applying nutritional ecology to optimize diets of crickets raised for food and feed

Increasing yield is a primary goal of mass insect rearing for food and feed, and diet shapes insect life history traits important to yield, such as survival, development time, and body size at adulthood. Little is known about how developmental macronutrient intake impacts survival, growth, and adult body size of mass reared insects. Here, we applied the nutritional geometry framework and reared individual tropical house crickets (Gryllodes sigillatus) from hatch to adulthood on a wide range of protein:carbohydrate diets. We measured weekly food consumption, survival, development time to adulthood, and adult body size and mass, and calculated a yield metric to extrapolate our individual-level results and predict how diet influences yield at the mass rearing level. Yield was maximized on a 3P:1C diet, as crickets fed this diet were most likely to develop into adults and grew maximum mass and body size. When provided with a choice between diets, crickets selected a relatively balanced 1.05P:1C diet throughout development, but males consumed 17% more protein than females. Our results represent a crucial first step towards determining the optimal standard feed formulation required to maximize cricket farming yield.

ecology↗

bioRxiv2024

Dual agonism of sodium iodide symporter function in vivo

New approaches are urgently needed to enhance the radioiodide (RAI) ablation of aggressive and metastatic thyroid cancer. We recently discovered that valosin-containing protein inhibitors (VCPi) such as clotrimazole and disulfiram transiently block sodium iodide symporter (NIS) proteasomal degradation, hence promoting RAI uptake. However, poor bioavailability diminishes their potential impact in vivo. Following 3D modelling and iterative drug design we appraised 26 novel analogues of clotrimazole, as well as albumin nano-encapsulated copper-diethyldithiocarbamate [Cu(DDC)2-alb] - a stabilised reformulation of a disulfiram metabolite. While several clotrimazole analogues specifically increased RAI uptake, the greatest impact was observed with Cu(DDC)2-alb in thyroid cancer cells as well as human primary thyrocytes from patients with thyroid hyperplasia. NanoBRET assays revealed that Cu(DDC)2 enhanced the plasma membrane accumulation of NIS in living cells. In BALB/c mice, both intraperitoneal and intravenous administration of Cu(DDC)2-alb significantly enhanced thyroidal 99mTc-uptake. RNA-Seq revealed the surprising observation that Cu(DDC)2-alb induced key thyroid transcription factors. Accordingly, expression of PAX8 and NKX2.1 was upregulated in thyroid glands from drug treated mice, with NIS levels correlating closely to 99mTc-uptake. As Cu(DDC)2 inhibits the VCP cofactor NPL4, with VCP being critical to the proteostatic processing of NIS protein, we identify a new dual agonist of RAI uptake in vivo, with the potential to directly impact RAI therapy for patients with aggressive thyroid cancer.

cancer biology↗

bioRxiv2023

Dissecting endocytic mechanisms reveals new molecular targets to enhance sodium iodide symporter activity with clinical relevance to radioiodide therapy

The sodium/iodide symporter (NIS) frequently shows diminished plasma membrane (PM) targeting in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide (RAI) treatment and poor prognosis. The mechanisms which govern the endocytosis of NIS away from the PM are ill-defined. Here, we challenged the hypothesis that new mechanistic understanding of NIS endocytosis would facilitate prediction of patient outcomes and enable specific drug modulation of RAI uptake in vivo. Through mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET, we identify an acidic dipeptide within the NIS C-terminus which mediates binding to the {sigma}2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. We propose that NIS internalisation is orchestrated by the interaction of a C-terminal diacidic motif with AP2{sigma}2, together with the proto-oncogene PBF acting via AP22. Given that NIS internalisation was specifically druggable in vivo, our data provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. SummaryWe delineate the role of endocytic genes in regulating NIS activity at the plasma membrane and highlight the potential for systemic targeting of endocytosis to enhance radioiodine effectiveness in radioiodine-refractory cancer cells.

cancer biology↗

bioRxiv2021

Natian and Ryabhatta--graphical user interfaces to create, analyze and visualize single-cell transcriptomic datasets

Single-cell transcriptomic analyses permit a high-resolution investigation of biological processes at the individual cell level. Single-cell transcriptomics technologies such as Drop-seq, Smart-seq, MARS-seq, sci-RNA-seq, and CELL-seq produce large volumes of data in the form of sequence reads. In general, the alignment of the reads to genomes and the enumeration of reads mapping to a specific gene results in a gene-count matrix. These gene-count matrix data require robust quality control and statistical analytical pipelines before data mining and interpretation. Among these post-alignment pipelines, the Seurat package in R is the most popular analytical pipeline for the analysis of single-cell data. This package provides quality control, normalization, principal component analysis, dimensional reduction, clustering, and marker identification among other functions needed to process and mine the single-cell transcriptomic data. While the Seurat package is continuously updated and includes a variety of functionalities, the user is still required to be proficient in the R programming language and its data structures to be able to execute the Seurat functions. Hence, there is a demand for a graphical user interface (GUI) that takes in relevant input information and processes the single-cell data using the Seurat pipeline. A GUI will also highly improve the access to single-cell data for life sciences researchers who are not trained in the command-line operation of the R platform. To meet this demand, we present R Shiny apps Natian and Ryabhatta to assist in the generation and analysis of Seurat files from a variety of different sources. The apps and example data can be downloaded from https://singlecelltranscriptomics.org. Natian allows users to create Seurat files from the output of multiple pipelines, integrate existing Seurat files, add metadata information, perform dimensional reduction analysis or upload dimensional reduction data, resume partially processed Seurat files and find cluster markers. Ryabhatta allows users to visualize gene expression using a variety of plotting options, analyze cluster markers, rename clusters, select cells from a graph or based on expression levels of markers, perform differential expression, count the number of cells in each condition, and perform pseudotime analysis using Monocle. We found that the use of these apps substantially improved the analytical and processing time and remove needless troubleshooting due to incompatible commands, typographical errors in scripts, and cluttering of the R environment with variables. We hope the use of these apps improves the use of single-cell data for life sciences research while also providing a tool to learn the functionalities of Seurat and R functions available for single-cell data analysis.

bioinformatics↗

bioRxiv2021

Maternal hyperglycemia impedes second heart field-derived cardiomyocyte differentiation to elevate the risk of congenital heart defects

Congenital heart disease (CHD) is the most prevalent structural malformations of the heart affecting [~]1% of live births. To date, both damaging genetic variations and adverse environmental exposure such as maternal diabetes have been found to cause CHD. Clinical studies show [~]fivefold higher risk of CHD in the offspring of mothers with pregestational diabetes. Maternal pregestational diabetes affects the gene regulatory networks key to proper cardiac development in the fetus. However, the cell-type specificity of these gene regulatory responses to maternal diabetes and their association with the observed cardiac defects in the fetuses remains unknown. To uncover the transcriptional responses to maternal diabetes in the early embryonic heart, we used an established murine model of pregestational diabetes. In this model, we have previously demonstrated an increased incidence of CHD. Here, we show maternal hyperglycemia (matHG) elicits diverse cellular responses during heart development by single-cell RNA-sequencing in embryonic hearts exposed to control and matHG environment. Through differential gene-expression and pseudotime trajectory analyses of this data, we identified changes in lineage specifying transcription factors, predominantly affecting Isl1+ second heart field progenitors and Tnnt2+ cardiomyocytes with matHG. Using in vivo cell-lineage tracing studies, we confirmed that matHG exposure leads to impaired second heart field-derived cardiomyocyte differentiation. Finally, this work identifies matHG-mediated transcriptional determinants in cardiac cell lineages elevate CHD risk and show perturbations in Isl1-dependent gene-regulatory network (Isl1-GRN) affect cardiomyocyte differentiation. Functional analysis of this GRN in cardiac progenitor cells will provide further mechanistic insights into matHG-induced severity of CHD associated with diabetic pregnancies.

developmental biology↗

bioRxiv2020

Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show that NO rescues calcification by a S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells (pAVICs) and single cell RNA-seq demonstrated regulation of NOTCH pathway by NO. A unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin proteasome pathway and implicated S-nitrosylation of USP9X in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated aortic valve disease and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a novel mechanism by which S-nitrosylation dependent regulation of ubiquitin-associated pathway prevents CAVD.

cell biology↗

bioRxiv2020

Fear Conditioning Potentiates the Hippocampal CA1 Commissural Pathway In vivo and Increases Awake Phase Sleep

The hippocampus is essential for spatial learning and memory. To assess learning we used contextual fear conditioning (cFC), where animals learn to associate a place with aversive events like foot-shocks. Candidate memory mechanisms for cFC are long-term potentiation and long-term depression, but there is little direct evidence of them operating in the hippocampus in vivo following cFC. Also, little is known about the behavioral state changes induced by cFC. To address these issues, we recorded local field potentials in freely behaving mice by stimulating in the left dorsal CA1 region and recording in the right dorsal CA1 region. Synaptic strength in the commissural pathway was monitored by measuring field excitatory postsynaptic potentials (fEPSPs) before and after cFC. After cFC, the commissural pathways synaptic strength was potentiated. Although recordings occurred during the wake phase of the light/dark cycle, the mice slept more in the post-conditioning period than in the pre-conditioning period. Relative to awake periods, in non-rapid eye movement (NREM) sleep the fEPSPs were larger in both pre- and post-conditioning periods. We also found a significant negative correlation between the animals speed and fEPSP size. Therefore, to avoid confounds in the fEFSP potentiation estimates, we controlled for speed-related and sleep-related fEPSP changes and still found that cFC induced long-term potentiation, but no significant long-term depression. Synaptic strength changes were not found in the control group that simply explored the fear-conditioning chamber, indicating that exploration of the novel place did not produce the measurable effects caused by cFC. These results show that following cFC, the CA1 commissural pathway is potentiated, likely contributing to the functional integration of the left and right hippocampi in fear memory consolidation. In addition, the cFC paradigm produces significant changes in an animals behavioral state, which are observable as proximal changes in sleep patterns.

neuroscience↗

bioRxiv2019

Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by enlargement of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the C-terminal EF-hand (CEF) domain. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stopgain variants that lead to loss of the CEF domain are stably expressed. However, stopgain variants show impaired localization suggesting a functional role for the CEF domain. The degradation of the MYL2-fs can be rescued by inhibiting the cells proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither MYL2-fs nor MYL2:p.Gly162Arg supports regular cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathies.\n\nAuthor SummaryWe report a novel frameshift variant in MYL2 that is associated with a severe form of infantile-onset hypertrophic cardiomyopathy. The impact of the variant is only observed in the recessive form of the disease in the proband and not in the parents who are carriers of the variant. This is in contrast to other dominant variants in MYL2 that are associated with cardiomyopathies. We compared the stability of this variant to that of other cardiomyopathy associated MYL2 variants and found molecular differences in the disease pathology. We also show different protein domain requirement for stability and localization of MYL2 in cardiomyocytes. Further, we used a fly model to demonstrate functional deficits due to the variant in the developing heart. Overall, our study shows a molecular mechanism by which loss-of-function variants in MYL2 are recessive while missense variants are dominant. We highlight the use of exome sequencing and functional testing to assist in the diagnosis of rare forms of diseases where pathogenicity of the variant is not obvious. The new tools we developed for in vitro functional study and the fly fluorescent reporter analysis will permit rapid analysis of MYL2 variants of unknown significance.

genetics↗