Biology subjects

Roy, S.

Publications and source records attributed to Roy, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2024

Functional study of Phaeodactylum tricornutum Seipin homolog highlights unique features of lipid droplets biogenesis in diatoms.

Diatoms are a major phylum of microalgae, playing crucial ecological roles. They derive from secondary endosymbiosis of a red alga by an unknown heterotrophic eukaryote, leading to a complex intracellular organization. In response to unfavorable conditions (stress), diatoms store oil in lipid droplets (LD), raising interest for applications, in particular biofuels. In spite of numerous investigations aiming to increase their oil content, LD biogenesis mechanisms in these organisms remain poorly understood. In this study, we functionally characterized the homolog of Seipin, a major actor of LD biogenesis, in the diatom Phaeodactylum tricornutum. PtSeipin shares conserved structural features with other Seipins, yet presents unique characteristics, that appear common to diatoms and more broadly Stramenopiles. We provide evidence that Stramenopiles Seipins were inherited from the host during secondary endosymbiosis. The localization of PtSeipin highlights that LD biogenesis can arise simultaneously from the endoplasmic reticulum (ER) and the plastids most external membrane. Finally, the knock-out of PtSeipin leads to a strong increase of TAG accumulation, a feature that is not observed in other organisms and is greatly enhanced following high light exposure. Our results suggest a redirection of lipid fluxes towards TAG synthesis, reduced TAG recycling or a combination of both.

plant biology↗

bioRxiv2024

Polyphenol-rich fraction of Bergenia ligulata (Wall.) Engl. sensitizes colon and breast cancer metastasis in vivo: Evidence of cell-type specific mechanism

BackgroundColon and breast cancer are one of the leading causes of mortality worldwide. Limited efficacies of present treatments highlighted an urgent need for improvement. Bergenia ligulata is known for its anticancer properties in Indian traditional and folk medicine though the molecular basis of its effects, particularly its anti-metastatic properties, is not well understood. Anti-prostate cancer activity of a LCMS defined polyphenol-rich fraction from Bergenia ligulata (PFBL) rhizome extract has already been published showing promising results in preclinical models. PurposeThe present study aims to explore anti-cancer activity and anti-metastatic potentials of PFBL against colon and breast cancers in both in vitro and preclinical settings. Study design and methodsPFBL alone or in synergy with standard chemotherapeutic drugs was tasted in CT26 and 4T1 subcutaneous solid tumors in BALB/c mice. The effect of PFBL was analyzed in terms of changes in tumor mass and different molecular marker levels. Anti-metastatic potential of PFBL was evaluated in CT26 and 4T1 lung metastasis model in mice focusing on the number of lung nodules and lung size. ResultsOur results evidenced that PFBL efficiently regressed both CT26 and 4T1-induced solid tumors alone and in combination with 5FU/doxorubicin without affecting the health of normal host. Notably, PFBL suppressed lung metastasis of 4T1 and CT26 cells in mice with great efficacy. Analysis of tumor and cell extracts suggested that colon cancer cells died by autophagy, while breast cancer cells majorly died by caspase-dependent apoptosis. PFBL actions involved AMPK-dependent inhibition of mTORC1 and subsequent increase in LC3II, PARP1, CDK4 and Cyclin D1 levels in both HCT116 and MCF7 cells; an elevation of intracellular ROS level was the major cause of death in both the cancer types. PFBL treatment also reversed the EMT marker expressions in in vivo and in vitro. ConclusionPFBL regressed colon and breast cancer metastasis through distinct mechanisms with little effect on healthy mice. The present study underscored PFBL as a novel anti-tumor and anti-metastasis candidate warranting further testing in clinical settings.

cancer biology↗

bioRxiv2024

Comparative Analysis of Lipid Nanoparticles in Pfizer-BioNTech and Moderna COVID-19 Vaccines: Insights from Molecular Dynamics Simulations

COVID-19 vaccines, such as Pfizer-BioNTechs BNT162b and Modernas mRNA-1273, have demonstrated robust efficacy. However, direct comparisons of their delivery vehicles remain limited. Notably, BNT162b requires storage at -80{degrees}C, while mRNA-1273 is stored at -20{degrees}C. This discrepancy in storage temperatures may be influenced by differences in the structure and stability of the lipid nanoparticles (LNPs) used in these vaccines. Ionizable lipids, such as SM-102 in Modernas vaccine and ALC-0315 in Pfizers vaccine, play a crucial role in LNP stability and function, affecting endosomal escape, cellular uptake, and drug release. Understanding these variations is essential for optimizing vaccine delivery systems. In our study, we use molecular dynamics simulations with the coarse-grained Martini forcefield to compare the LNPs in Moderna and Pfizers COVID-19 vaccines, providing insights at an experimental scale. Our findings indicate that the ionizable lipid tail of BNT162b (ALC-0315) exhibits a higher degree of branching, resulting in a more bifurcated appearance compared to the structure of the ionizable lipids in mRNA-1273 (SM-102).

biophysics↗

bioRxiv2024

In Silico Engineering of Stable siRNA Lipid Nanoparticles: Exploring the Impact of Ionizable Lipid Concentrations for Enhanced Formulation Stability

Lipid nanoparticles (LNPs) are crucial in advancing the delivery of RNA-based therapeutics within the domain of gene therapy. A comprehensive understanding of their formation and stability is critical for optimizing the clinical efficacy of LNPs. This study systematically investigates the influence of concentration variations of positive and neutral ionizable lipids - specifically, 2-[2,2-bis[(9Z,12Z)-octadeca-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethanamine (DLinKC2-DMA) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) - along with cholesterol and polyethylene glycol, on the formation of LNPs and encapsulation of small interfering RNA (siRNA). Utilizing coarse-grained classical molecular dynamics (MD) simulations with a system size matching experimental range (approximately 0.6 million beads), we conduct a comparative analysis and offer mechanistic insights into siRNA formulation within LNPs containing positive and neutral DLinKC2-DMA. We found that the LNPs with positive ionizable lipids encapsulate more than twice the siRNA compared to the LNPs with neutral ionizable lipids. In addition to the formation of LNPs, our study extends to the forces governing siRNA escape from LNPs, employing steered molecular dynamics simulations. The force experienced by siRNA to cross the LNP lipid layer containing positive ionizable lipids was 400kJ/mol/nm more than that of neutral ionizable lipids, suggesting the encapsulation is more favorable with positive ionisable lipids.

biophysics↗

bioRxiv2024

Cytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems

CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of published and new CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection bottlenecks that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.

molecular biology↗

bioRxiv2024

Diet induced mitochondrial DNA replication instability in Rad51c mutant mice drives sex-bias in anemia of inflammation

Anemia of inflammation (AI) is a common comorbidity associated with obesity, diabetes, cardiac disease, aging, and during anti-cancer therapies. Mounting evidence illustrates that males are disproportionally affected by AI, but not why. Here we demonstrate a molecular cause for a sex-bias in inflammation. The data shows that mitochondrial DNA (mtDNA) instability induced by dietary stress causes anemia associated with inflamed macrophages and improper iron recycling in mice. These phenotypes are enhanced in mice with mutations in Fanco/Rad51c, which predisposes to the progeroid disease Fanconi Anemia. The data reveals a striking sex-bias whereby females are protected. We find that estrogen acts as a mitochondrial antioxidant that reduces diet-induced oxidative stress, mtDNA replication instability and the distinctively mtDNA-dependent unphosphorylated STAT1 response. Consequently, treatment of male Rad51c mutant mice with estrogen or mitochondrial antioxidants suppresses the inflammation-induced anemia. Collectively, this study uncovers estrogen-responsive mtDNA replication instability as a cause for sex-specific inflammatory responses and molecular driver for AI.

molecular biology↗

bioRxiv2024

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes

Aims/hypothesisSurviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. MethodsBeta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. ResultsPD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN- or IFN-{square} treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretationIFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death. Research in contextO_ST_ABSWhat is already known about this subject? (maximum of 3 bullet points)C_ST_ABSO_LIExtracellular vesicles (EVs) serve as paracrine effectors in the islet microenvironment in health and disease. C_LIO_LIInterferon-alpha (IFN-) and IFN-gamma (IFN-{square}) are key cytokines contributing to type 1 diabetes pathophysiology and islet IFN signalling increases beta cell programmed death-ligand 1 (PD-L1) expression. C_LIO_LIUp-regulation of beta cell PD-L1 in the non-obese diabetic (NOD) mouse model delays progression of type 1 diabetes. C_LI What is the key question? (one bullet point only; formatted as a question)O_LIDo beta cells exposed to IFNs upregulate EV PD-L1 and can these changes be detected in circulation? C_LI What are the new findings? (maximum of 3 bullet points)O_LIIFN- or IFN-{square} exposure increases beta cell EV PD-L1 cargo in beta cell lines and human islets. C_LIO_LIPD-L1 is present on the surface of beta cell EVs, binds PD-1 and EV PD-L1 inhibits proliferation, activation and cytotoxicity of murine CD8 T cells. C_LIO_LIEV PD-L1 levels are higher in islet autoantibody positive individuals compared to nondiabetic controls and levels of circulating EV PD-L1 positively correlate with residual beta cell function in islet autoantibody positive individuals as well as in individuals with recent-onset type 1 diabetes. C_LI How might this impact on clinical practice in the foreseeable future? (one bullet point only)O_LIA beneficial effect of PD-L1+ EVs could ultimately be harnessed as an intervention to prevent autoimmune beta cell destruction. Circulating EV PD-L1 cargo has potential as a minimally invasive and informative biomarker to offer insights into the pathogenesis and progression of type 1 diabetes. C_LI

cell biology↗

bioRxiv2024

Modulating Prefrontal Cortex Activity to Alleviate Stress-Induced Working Memory Deficits: A Transcranial Direct Current (tDCS) Study

This study explores the impact of stress on working memory (WM) performance, and the potential mitigating effects of transcranial direct current stimulation (tDCS) over the left dorsolateral prefrontal cortex (dlPFC) and ventromedial prefrontal cortex (vmPFC). The study had a crossover, randomized, single-blind, sham-controlled design, with stress induction as within-subject and stimulation condition as between-subject factors. We assessed stress-induced WM deficits using aversive video clips to induce stress and a verbal n-back task to assess WM performance. We analyzed physiological (cortisol and heart rate), behavioral, and electroencephalographic (EEG) changes due to stress before, during, and after WM task performance and their modulation by tDCS. Stress impaired WM performance in the sham stimulation condition for the 3-back load, but not for 2-back or 4-back loads in the WM task, and was associated with elevated physiological stress markers. tDCS over the vmPFC led to better WM task performance while stimulation over the dlPFC did not. Active tDCS with both dlPFC and vmPFC stimulation blunted cortisol release in stress conditions compared to sham. The EEG analysis revealed potential mechanisms explaining the behavioral effects of vmPFC stimulation. vmPFC stimulation led to a decreased P200 event-related potential (ERP) component compared to the sham stimulation condition and resulted in higher task-related alpha desynchronization, indicating reduced distractions and better focus during task performance. This study thus shows that the vmPFC might be a potential target for mitigating the effects of stress on WM performance, and contributes to the development of targeted interventions for stress-related cognitive impairments. Significance StatementThis study investigates how stress impacts working memory performance and whether non-invasive brain stimulation can mitigate these effects. Using transcranial direct current stimulation (tDCS), we targeted specific brain areas in participants stressed by watching aversive videos. Stress reduced working memory performance and increased cortisol levels. However, tDCS applied to the ventromedial prefrontal cortex (vmPFC) improved task performance and blunted the cortisol response compared to a sham treatment. EEG data suggest that stimulation also enhanced focus during task performance and reduced emotional distraction. Our findings suggest that tDCS over the vmPFC could be a promising non-invasive brain stimulation method to counteract the negative effects of stress on cognitive functions.

neuroscience↗

bioRxiv2024

CUP-SHAPED COTYLEDON2 activates MIR319C transcription and promotes cell proliferation in Arabidopsis leaf primordia

The microRNA miR319 regulates leaf size in diverse plant species by reducing the level of the target transcripts that encode JAW-TCPs, the transcription factors (TF) that restrict leaf size by committing the proliferating pavement cells to differentiation. MIR319C, one of the three miR319-producing genes in Arabidopsis, is expressed throughout the incipient leaf primordia, and its expression domain gets restricted to the base at later stages, partly due to its transcriptional repression by JAW-TCPs. However, the factors that activate and maintain MIR319C expression in leaf primordia are yet unknown. Here, we identify the CUP-SHAPED COTYLEDON2 (CUC2) TF as a direct activator of MIR319C transcription. Using a yeast one-hybrid (Y1H) screen, we identified several NAC domain TFs as potential regulators of MIR319C. Subsequent ex vivo binding and transactivation assays suggested that CUC2 binds to a distal promoter region of the MIR319C locus and activates its transcription. Mutants with compromised CUC2 and MIR319C activities resulted in smaller leaves with fewer cells. Detailed morphometric analysis of higher order CUC2 and MIR319 loss-of-function mutants highlighted the crucial role of the CUC2-MIR319 module in maintaining the duration of cell proliferation in leaf primordia. Additionally, the phenotype of mutants with altered CUC2 and MIR319/JAW-TCP activities demonstrated that CUC2 enhances leaf size through the MIR319C-JAW-TCP pathway. Overall, our findings uncovered a novel role for CUC2 in sustaining cell division by activating MIR319C transcription in the leaf primordia.

plant biology↗

bioRxiv2024

A conserved cell-type gradient across the human mediodorsal and paraventricular thalamus

The mediodorsal thalamus (MD) and adjacent midline nuclei are important for cognition and mental illness, but their cellular composition is not well defined. Using single-nucleus and spatial transcriptomics, we identified a conserved excitatory neuron gradient, with distinct spatial mapping of individual clusters. One end of the gradient was expanded in human MD compared to mice, which may be related to the expansion of granular prefrontal cortex in hominids. Moreover, neurons preferentially mapping onto the parvocellular division MD were associated with genetic risk for schizophrenia and bipolar disorder. Midbrain-derived inhibitory interneurons were enriched in human MD and implicated in genetic risk for major depressive disorder.

neuroscience↗

bioRxiv2024

Estrogen receptor alpha (ERα) driven trans-regulation of mitotic checkpoint complex (MCC) components affects the clinical outcome of breast cancer

Hormone receptors (HR), namely estrogen receptor (ER) and progesterone receptor (PR), are prevalent in most malignant tumors. Although previous literature provided clues for ER in regulating mitosis and ploidy status in breast cancer (BC) cells, reports on the mitotic regulators being the targets of HR are sparse. To delve deeper into ERs impact on mitotic execution, our study focuses on examining its transcriptional activity on the core mitotic checkpoint complex (MCC) components, which are involved in ploidy maintenance. The expression of the core MCC components (Bub3, Mad2, and BubR1) was analyzed by quantitative-PCR and immunohistochemistry in breast tumors and adjacent normal tissues from the cancer genome atlas-breast invasive carcinoma collection (TCGA BRCA) dataset and in a prospective cohort of Eastern Indian breast cancer affected individuals. The preliminary data from these cohorts indicated an influence of ER on the two MCC components, namely Mad2 and BubR1. Subsequently, luciferase reporter assays and chromatin immunoprecipitation were performed which revealed that ER promotes transcriptional activation of MAD2 and BUB1B through direct recruitment on these promoters, showing affects in mitotic outcome. Interestingly, the ectopic introduction of ER, in an HR-ve breast cancer line, MDA-MB-231, significantly reduced the percent aneuploidy. Moreover, we found that overexpression of MAD2 and BUB1B is associated with poorer survival in HR-positive (HR+ve) patients in both cohorts. Our findings provide insights into the specific role of ER-mediated transcriptional regulation of mitosis and ploidy outcome. Targeting the deregulated MCC components thus offers translational potential for the therapeutic management of breast cancer.

cancer biology↗

bioRxiv2024

Predicting small-molecule inhibition of protein complexes

MotivationProtein-Protein Interactions (PPIs) are crucial in biological processes and disease mechanisms, underscoring the importance of discovering PPI inhibitors in drug development. Machine learning can expedite this discovery process. Although machine learning techniques for predicting general compound inhibition are available, we are not aware of any that accurately forecast the inhibitory effect of a compound on a specific protein complex, utilizing inputs from both the compound and the protein complex. MethodsWe present the first targeted machine learning based predictor of small molecule based inhibition of protein complexes. Our proposed graph neural network integrates the structure of a protein complex, its protein-protein binding site or interface features and a compounds SMILES representation to predict the potential of the given compound to inhibit the interaction between proteins in the given complex in a targeted manner. ResultsValidated on the 2p2i-DB-v2 database, encompassing 714 inhibitors across 23 complexes with over 12,000 instances, our model achieves superior predictive accuracy (cross-validation AUC-ROC of 0.86), outperforming established kernel methods and pre-trained neural networks. We further tested the predictive performance of our model on two independent external datasets - one collected from recent publications and another consisting of putative inhibitors of the SARS-CoV-2-Spike and Human-ACE2 protein complex with AUC-ROCs of 0.82 and 0.78, respectively. Our targeted predictor introduces a novel approach for PPI inhibitor discovery, laying foundational work for future advancements in addressing this complex and previously unexplored prediction challenge. AvailabilityCode/supplementary material available: https://github.com/adibayaseen/PPI-Inhibitors

bioinformatics↗

bioRxiv2024

An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

Several studies have reported the importance of aerobic glycolysis in melanoma development. Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in melanoma growth and proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as GAPDH and PKM2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis, is an RNA binding protein (RBP) that regulates mRNA translation in melanoma. We show that siRNA-mediated HK2 depletion changes the translational landscape of melanoma cells. Polysome profiling experiments and RNA-Seq indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates the translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance and progression in melanoma. RNA-protein interaction assays, including crosslinking immunoprecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its hexokinase activity or subcellular localization. We also show that HK2 specifically associates with the 5 untranslated region (5UTR) of the SOX10 mRNA, and that several deletions in this region decreases both HK2-SOX10 mRNA association and SOX10 5 UTR-mediated translation. We further show that HK2-dependent SOX10 translational regulation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a non-metabolic function of HK2, indicating that melanoma cells may enhance glycolysis for purposes beyond simple anabolism.

cancer biology↗

bioRxiv2024

The prion-like protein Doppel: A soluble biomarker steering ovarian cancers peritoneal to circulatory dissemination

Detecting ovarian cancer (OC) early using existing biomarkers, e.g., cancer antigen 125 (CA125), is challenging due to its ubiquitous expression in many tissues. Doppel, a prion-like protein, expresses in male reproductive organ but absent in female reproductive systems and healthy tissues, but plays an important role in neoangiogenesis. Here, we have shown two platforms, soluble Doppel in sera/ascites and Doppel expressed circulating tumor cells (Dpl+CTC) in the whole blood, to detect subsets of epithelial OC (EOC). Increased level of Doppel in the sera of OC patients, in three different cohorts, confirm Doppel as OC specific biomarker. Serum Doppel level distinguishes EOC subtypes and early stages HGSOCs from non-cancerous conditions with high sensitivity and specificity. Stratifying the EOCs based on Doppel level, we categorized them into Doppel-high (Dplhi) and Doppel-low (Dpllow) groups. Using ascites-derived organoids and single cell sequencing of whole ascites of Dplhi and Dpllow patients, we identify that Doppel induces epithelial-mesenchymal transition (EMT) and creates an immunosuppressive microenvironment, respectively. Doppel levels in the sera/ascites correlate with the changes of Dpl+CTC number in whole blood, highlighting the association of Doppel-induced EMT with CTC dissemination in circulation. Thus, Doppel-based detection of EOC subtypes could be a promising platform as clinical biomarker and link Doppel-axis with OC dissemination.

cancer biology↗

bioRxiv2024

DGTS overproduced in seed plants is excluded from plastid membranes and promotes endomembrane expansion

Plants and algae must adapt to environmental changes, facing various stresses that negatively impact their growth and development. One common stress is phosphate (Pi) deficiency, which is often in limiting quantity in the environment. In response to Pi deficiency, these organisms increase Pi uptake and remobilize intracellular Pi. Phospholipids are degraded to provide Pi and replaced by non-phosphorous lipids, such as glycolipids or betaine lipids. During the evolution, seed plants lost their capacity to synthesize betaine lipid. By expressing BTA1 genes, involved in the synthesis of diacylglyceryl-N,N,N-trimethyl-homoserine (DGTS), from different species, our work shows that DGTS can be produced in seed plants. In Arabidopsis, expressing BTA1 under a phosphate starvation-inducible promoter resulted in limited DGTS production without having any impact on plant growth or lipid remodeling. In transient expression systems in Nicotiana benthamiana, leaves were able to accumulate DGTS up to 20 % of their glycerolipid content at a slight expense of galactolipid and phospholipid production. At the subcellular level, we showed that DGTS is absent from plastid and seems to be enriched in endomembrane, driving an ER membrane proliferation. Finally, DGTS synthesis pathway seems to compete with PC synthesis via the Kennedy pathway but does not seem to be derived from PC diacylglycerol backbone and therefore does not interfere with the eukaryotic pathway involved in galactolipid synthesis.

plant biology↗

bioRxiv2024

Early 2022 breakthrough infection sera from India target the conserved cryptic class 5 epitope to counteract immune escape by SARS-CoV-2 variants

During the COVID-19 pandemic, the vast majority of epitope mapping studies have focused on sera from mRNA-vaccinated populations from high-income countries. In contrast, here we report an analysis of 164 serum samples isolated from breakthrough infection patients in India during early 2022 who received two doses of the ChAdOx viral vector vaccine. Sera were screened for neutralization breadth against wildtype, Kappa, Delta, and Omicron BA.1 viruses. Three sera with the highest neutralization breadth and potency were selected for epitope mapping using charged scanning mutagenesis coupled to yeast surface display and Next Generation Sequencing. All of these sera primarily targeted the recently identified class 5 cryptic epitope along with the class 1 and class 4 epitopes, targeted to a lesser extent. The class 5 epitope is completely conserved across all SARS-CoV-2 variants and for the majority of the Sarbecoviruses. In line with these observations, a major fraction of the serum samples, including the selected three, show broad neutralizing activity against recent Omicron subvariants including XBB.1.5. This is in contrast with the results obtained with the sera from individuals receiving multiple doses of original and updated mRNA vaccines, where impaired neutralization of XBB and later variant of concerns was observed. Our study demonstrates that two doses of the ChAdOx vaccine in a highly exposed population was sufficient to drive substantial neutralization breadth against emerging and upcoming variants of concern, thus paves the path towards the development of future vaccine candidates. ImportanceWorldwide implementation of COVID19 vaccine and parallel emergence of newer SARS-CoV-2 variants has shaped the humoral immune response in a population specific manner. While characterization of this immune response is important for monitoring the disease progression at population level, it is also imperative for the development of effective countermeasures in the form of novel vaccines and therapeutics. India has implemented the worlds second largest COVID19 vaccination drive and also encountered large number of post vaccination "breakthrough" infections. From a cohort of breakthrough infection patients, we identified individuals whose sera showed broadly cross-reactive immunity against different SARS-CoV-2 variants. Interestingly, these sera primarily target a novel cryptic epitope which was not identified in previous population level studies conducted in western countries. This rare cryptic epitope remains conserved across all SARS-CoV-2 variants including the recently emerged ones and also for the SARS-like coronaviruses that may cause future outbreaks, thus representing a potential target for future vaccines.

molecular biology↗

bioRxiv2024

TGFb-ANGPT2-Tie2 axis in cancer-associated fibroblasts reprograms oral cancer cells to embryonic-like cell state with predictive significance of poor prognosis

Myofibroblastic cancer-associated fibroblasts (CAFs) in tumor stroma is identified as poor-prognostic indicator in oral cancer; however, biological mechanisms are largely unexplored. Here, we discovered the role of autocrine or exogenous transforming growth factor beta (TGF{beta}) in inducing Tunica Interna Endothelial cell kinase 2 (Tie2) -signaling through histone deacetylase-mediated downregulation of Tie2-antagonist, Angiopoietin-2 in CAFs, responsible for induction and maintenance of myofibroblastic differentiation. To understand the influence of CAF-specific Tie2-signaling on cancer cell properties, we performed CAF-Cancer cell co-culture and its single-cell RNA sequencing (scRNA-Seq). Distinct clustering of CAFs suggested their transcriptional heterogeneity, driven by TGF{beta}-Tie2 activation. Interestingly, CAF-specific Tie2-signaling was responsible to reprogram cancer cells, producing embryonic-like cell state with increased stemness and EMT signatures. Importantly, both the Tie2-specific gene expression signature as well as reprogrammed cancer cell specific gene expression modules were validated respectively in fibroblasts clusters and malignant cell clusters in two independent earlier reported scRNAseq studies of HNSCC tumors. Highlighting the translatability of our study, the gene expression signature derived from reprogrammed cancer cells showed significant association with poor prognosis in HNSCC patient of TCGA cohort. Pharmacological inhibition of Tie2-signaling in CAFs, significantly abrogated the tumor initiating ability of co-cultured oral cancer cell lines. Overall, combining our molecular and computational analysis, we may propose Tie2 as a novel factor responsible for CAF mediated cancer cell plasticity, associated with aggressive nature of oral cancer. TeaserTie2-signaling is activated in myofibroblasts which impacts the behaviour of malignant cells by inducing cancer cell plasticity to acquire stemness.

cancer biology↗

bioRxiv2024

MICROBIOME-MODIFIED METABOLITES PREDICT ACUTE BRAIN INJURY OUTCOME

BACKGROUNDAcute brain injury (ABI)-mediated disruption of the microbiome may potentiate inflammation and secondary brain injury (SBI). However, microbial-specific mediators and mechanisms remain unclear. METHODSThirty-five consecutive patients with ABI admitted to the neuroscience critical care unit at the University of Chicago were prospectively studied. Injury severity at hospital admission was assessed using the Injury Severity Score (ISS) and the Glasgow Coma Scale (GCS). Final neurologic function was assessed via the Glasgow Outcome Score extended (GOSe). Serum, plasma, and stool targeted metabolomics, as well as stool shotgun metagenomics, were performed on longitudinal samples collected during hospitalization. RESULTSMultivariate analysis identified microbiome-modified metabolites that were positively and negatively associated with functional outcomes after ABI. Novel identification of conjugated bile acid (BA) species and vitamin B12 precursors indicative of outcome were detected in the first collected samples (within 48 hours). Network analysis revealed greater integration of negatively associated metabolites across tissues and identified tauro-/-muricholic acid (TMCA) as central to the cross-tissue metabolomes. CONCLUSIONSMicrobiome metabolites may be useful in assessing brain injury outcomes to inform treatment. Bile acid species transformed by the gut microbiome are predictive of ABI outcome.

neuroscience↗