Biology subjects

Iyer, S.

Publications and source records attributed to Iyer, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Identification of markers for the isolation of neuron-specific extracellular vesicles

Extracellular vesicles (EVs) are released by all cells and contain RNA and protein from their cell of origin. EVs in biofluids could be used as diagnostic biomarkers to non-invasively report the state of inaccessible cells, such as neurons in the brain. As biofluids such as cerebrospinal fluid (CSF) and plasma contain EVs originating from many different cells, isolating cell type-specific EVs and measuring their cargo could help determine the state of specific cell types. Here, we demonstrate an approach aiming to immuno-isolate EVs from neurons based on neuron-derived protein surface markers. We first developed a framework to select transmembrane proteins suitable as neuron-specific EV markers based on gene expression and EV proteomics data. Leveraging a novel, high-purity EV isolation method we developed, we further cataloged the proteins present on EVs in human CSF and plasma. Using ultrasensitive immunoassays against several of the predicted neuron-specific proteins, we confirmed one marker, NRXN3 as present on EVs in CSF and plasma by size exclusion chromatography (SEC) and density gradient centrifugation (DGC). Finally, we developed efficient EV immuno-isolation methods and applied them to isolate NRXN3+ EVs. Our study provides a general methodology for the isolation of cell-type specific EVs and paves the way for the use of neuron-derived EVs to study and diagnose neurological disease.

molecular biology↗

bioRxiv2024

Impaired AMPK Control of Alveolar Epithelial Cell Metabolism Promotes Pulmonary Fibrosis

Alveolar epithelial type II (AT2) cell dysfunction is implicated in the pathogenesis of familial and sporadic idiopathic pulmonary fibrosis (IPF). We previously described that expression of an AT2 cell exclusive disease-associated protein isoform (SP-CI73T) in murine and patient-specific induced pluripotent stem cell (iPSC)-derived AT2 cells leads to a block in late macroautophagy and promotes time-dependent mitochondrial impairments; however, how a metabolically dysfunctional AT2 cell results in fibrosis remains elusive. Here using murine and human iPSC-derived AT2 cell models expressing SP-CI73T, we characterize the molecular mechanisms governing alterations in AT2 cell metabolism that lead to increased glycolysis, decreased mitochondrial biogenesis, disrupted fatty acid oxidation, accumulation of impaired mitochondria, and diminished AT2 cell progenitor capacity manifesting as reduced AT2 self-renewal and accumulation of transitional epithelial cells. We identify deficient AMP-kinase signaling as a key upstream signaling hub driving disease in these dysfunctional AT2 cells and augment this pathway to restore alveolar epithelial metabolic function, thus successfully alleviating lung fibrosis in vivo.

molecular biology↗

bioRxiv2023

Calcium Sulfate Microparticle Size Modification for Improved Alginate Hydrogel Fabrication and Its Application in 3D Cell Culture

Calcium ion-crosslinked alginate hydrogels are widely used as a materials system for investigating cell behavior in 3D environments in vitro. Suspensions of calcium sulfate particles are often used as the source of Ca2+ to control the rate of gelation. However, the instability of calcium sulfate suspensions can increase chances of reduced homogeneity of the resulting gel and requires researchers proficiency. Here, we show that ball-milled calcium sulfate microparticles with smaller sizes can create more stable crosslinker suspensions than unprocessed or simply autoclaved calcium sulfate particles. In particular, 15 {micro}m ball-milled calcium sulfate microparticles result in gels that are more homogeneous with a balanced gelation rate, which facilitates fabrication of gels with consistent mechanical properties and reliable performance for 3D cell culture. Overall, these microparticles represent an improved method for alginate hydrogel fabrication that can increase experimental reliability and quality for 3D cell culture.

bioengineering↗

bioRxiv2023

Measuring, visualizing and diagnosing reference bias with biastools

Many bioinformatics methods seek to reduce reference bias, but no methods exist to comprehensively measure it. Biastools analyzes and categorizes instances of reference bias. It works in various scenarios, i.e. (a) when the donors variants are known and reads are simulated, (b) when donor variants are known and reads are real, and (c) when variants are unknown and reads are real. Using biastools, we observe that more inclusive graph genomes result in fewer biased sites. We find that end-to-end alignment reduces bias at indels relative to local aligners. Finally, we use biastools to characterize how T2T references improve large-scale bias.

bioinformatics↗

bioRxiv2023

Robust acidic pH and digestive enzyme stability of anti-SarsCov2 IgY antibodies: Implications for treatment of viral transmission thru gastrointestinal, ocular, nasal and skin tissues

There are many paths for the transmission of SarsCov2 virus. The main routes are nasal and oral and the droplets carrying the virus can also be transmitted thru ocular and skin tissues. The gastrointestinal (small intestine), nasal, ocular and skin tissues all present an acidic pH milieu and therefore any treatment with antibodies thru these routes has to have the antibodies remain active at acid pH as well as be resistant to typical protease digestion. To this end we profiled our anti-SarasCov2 receptor binding domain IgY antibodies for retention of activity at acidic and basic pHs and trypsin digestion. We find that the IgY are strongly resistant to denaturation at acid pH as well as not digested by trypsin. Our data strongly support the use of these IgY in treatment of viral transmission thru the GI, nasal, ocular and skin all tissues where the pH is acidic. We also provide an enabling platform to rapidly asses the suitability of any antibody or protein therapeutic for use at acidic pH.

pharmacology and toxicology↗

bioRxiv2023

The T766M-EGFR lung cancer mutation promotes tumor growth by exploiting newfound assembly mechanisms in ligand-free EGFR oligomers

Epidermal growth factor receptor (EGFR) is central to cell growth in physiology and pathophysiologies, including non-small cell lung cancer (NSCLC). EGFR has been successfully targeted with tyrosine kinase inhibitor generations, but the missense secondary T766M mutation is a common cause of resistance. Overcoming this therapeutic challenge has been hindered by poor understanding of how T766M dysregulates EGFR function leading to tumor progression. Here we show that T766M amplifies tumor growth in vivo by exploiting newly discovered oligomer assembly mechanisms employed by wild type (WT)-EGFR to maintain ligand-independent basal phosphorylation. These mechanisms, also shared by drug-resistant exon 20 EGFR insertions, reveal tumor growth promoting functions for hitherto orphan transmembrane and kinase interfaces and for the ectodomain tethered conformation of EGFR. Placing our findings into the context of a ligand-free oligomer structure model, we provide a framework for future drug discovery directed at tackling EGFR mutations in cancer by disabling oligomer-assembling interactions.

cancer biology↗

bioRxiv2023

Disruption of Prostaglandin F2α Receptor Signaling Attenuates Fibrotic Remodeling and Alters Fibroblast Population Dynamics in A Preclinical Murine Model of Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2, and its cognate receptor FPr (Ptfgr) are implicated as a TGF{beta}1 independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER -SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen treated IER -Sftpc I73T mice develop an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER -Sftpc I73T mice crossed to a Ptgfr null (FPr-/-) line showed attenuated weight loss and gene dosage dependent rescue of mortality compared to FPr+/+ cohorts. IER -Sftpc I73T /FPr-/- mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single cell RNA sequencing, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts which were reprogrammed to an "inflammatory/transitional" cell state in a PGF2/ FPr dependent manner. Collectively, the findings provide evidence for a role for PGF2 signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.

molecular biology↗

bioRxiv2023

Targeted neutralizing IgY antibodies against SGLT1 glucose transporter reduce glucose uptake and improve glycemic profile in vivo

Despite the use of several drugs available to treat type 2 diabetes, many patients are unable to reach their target fasting plasma glucose and HbA1c levels. SGLT1 is the major intestinal transport transmembrane protein which functions in uptake of dietary glucose. If we antagonise the binding of dietary glucose to this transport protein, it is expected that blood glucose lowering will follow. We designed specific inhibitory avian antibodies (IgY) against the extracellular glucose binding domain of SGLT1 and tested their potential in glucose lowering. We demonstrate here the antibodies block uptake of glucose and improve the glycemic profile in vivo and represent a novel approach to inhibiting dietary glucose absorption as treatment for diabetes.

pharmacology and toxicology↗

bioRxiv2023

An Anti-diabetic Nutraceutical enhances muscle cell glucose uptake of cardiometabolic drugs

Type 2 diabetes is currently treated by multiple drugs that are often combined to achieve maximum blood glucose lowering in patients. Yet more than 50% of patients are unable to attain target glucose levels. There is clear need for agents which can be added to current drugs to help patients achieve their target blood glucose levels safely. To this end we tested an anti-diabetic nutraceutical for its ability to enhance glucose uptake in muscle cells in combination with currently used cardiometabolic drugs. We show here that the nutraceutical is able to effectively improve glucose uptake of multiple drugs suggesting that it may help in enhancement of glucose lowering by the drugs in patients and achieve optimal glucose levels.

pharmacology and toxicology↗

bioRxiv2023

Negative Affect homogenizes and Positive Affect diversifies Social Memory Consolidation across people

We are often surprised when an interaction we remember positively is recalled by a peer negatively. What colors social memories with positive versus negative hues? We show that when resting after a social experience, individuals showing similar default network responding subsequently remember more negative information, while individuals showing idiosyncratic default network responding remember more positive information. Results were specific to rest after the social experience (as opposed to before or during the social experience, or rest after a nonsocial experience). The results provide novel neural evidence in support of the "broaden and build" theory of positive emotion, which posits that while negative affect confines, positive affect broadens idiosyncrasy in cognitive processing. For the first time, we identified post-encoding rest as a key moment and the default network as a key brain system in which negative affect homogenizes, whereas positive affect diversifies social memories.

neuroscience↗

bioRxiv2023

The Spread of Interferon-γ in Melanomas is Highly Spatially Confined, Driving Non-Genetic Variability in Tumor Cells

Interferon-{gamma} (IFN{gamma}) is a critical anti-tumor cytokine that has varied effects on different cell types. The global effect of IFN{gamma} in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFN{gamma} spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFN{gamma} in melanomas. We observe spatially confined niches of IFN{gamma} signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFN{gamma} signaling only occurs when niches overlap due to high local densities of IFN{gamma}-producing T cells. We measured length scales of [~]30-40m for IFN{gamma} spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFN{gamma} spread being governed by a simple diffusion-consumption model, and offer insight into how the spatial organization of T cells contributes to intra-tumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": our work provides a general explanation for such non-genetic cellular variability due to confinement in the spread of immune mediators.

systems biology↗

bioRxiv2023

Profiling extracellular vesicles in circulation enables the early detection of ovarian cancer

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53, and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study (n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

cancer biology↗

bioRxiv2022

Salivary Glands treated with AAV vectors encoding PYY and exendin-4 impact upon taste perception and body mass accumulation in mice.

The availability of high-calorie foods is likely a causative factor for high rates of obesity and metabolic disorders, which have been linked to food intake dysregulation. Several gut peptides have been implicated in feeding modulation and body mass accumulation. For example, glucagon peptide-like 1 (GLP-1) and peptide tyrosinetyrosine (PYY) have been shown to mediate satiety and reduce food intake. While systemic administration of such peptides has been explored as a therapy for metabolic disease, the effects of these hormones on taste signaling should also be considered given the importance of taste to feeding decisions. Peptide signaling systems are present in taste buds and oral GLP-1 and PYY signaling has been shown to influence taste responsiveness and feeding. Indeed, we previously demonstrated that genetic knockout of PYY in mice can impact on taste responsiveness and feeding and that viral overexpression of PYY in the salivary glands of these mice can rescue responsiveness. The present work uses AAV-mediated salivary gland treatment of both GLP-1 receptor agonist exendin-4 and PYY encoding vectors to explore the impact of the presence of these peptides on taste and body-mass accumulation in wild-type mice with intact peptide signaling systems. Results showed a significant effect of salivary gland treatment on responsiveness to multiple taste qualities. Treatment with a vector designed to overexpress both peptides in saliva resulted in substantial reduction in body mass accumulation. These findings show taste modulation and impacts on body mass accumulation by the targeting of salivary glands with vectors designed to overexpress metabolic peptides in wild-type mice and suggest that the taste bud is a promising substrate for food intake modulation.

neuroscience↗

bioRxiv2022

A replication study on key neurodevelopmental pathways affected by valproate treatment ofneural precursor cells

Adults with bipolar disorder or epileptic seizures are commonly prescribed sodium valproate. In utero exposure to this drug is linked to a multitude of defects in normal brain development, from neural tube defects to autism spectrum disorders. During the course of brain development, neural precursor cells (NPCs) give rise to neurons and glia, and therefore to understand the valproate-induced defects, it is crucial to understand its effect on NPCs. Two NPC lines, both derived from healthy individuals, were used for all experiments. Cells were treated with 0.7mM valproate for one week. Fresh media (+/- drug) was replenished every alternate day. RNA was extracted on day 7 of drug treatment, and transcriptomics performed. All experiments were performed in biological replicates. Genes that showed >1-fold difference (with FDR adjusted q-value [≤] 0.05) were considered differentially expressed. We further investigated the interacting partners of the differentially expressed genes using PINOT, as well as cellular pathways using DAVID. Our primary endpoint of analysis were genes that were differentially expressed (DEGs) with valproate treatment in both the NPC lines used. We found 21 such genes that were common in the two lines. PINOT revealed 504 interacting partners of the DEGs. Functional annotation analysis showed significant enrichment of four signaling pathways - Wnt, Notch, Rho-GTPase and PI3K-AKT. While the role of Rho-GTPase is a novel finding, we have replicated previously reported findings on Wnt, Notch and PI3K-Akt pathways, which further strengthens their role in mediating neurodevelopmental anomalies.

neuroscience↗

bioRxiv2022

Suppression of insulin stimulated muscle glucose uptake by non-caloric sweetener sucralose and its reversal by an antidiabetic nutraceutical supplement

Non caloric sweeteners (NCS) have been used for decades now as sugar substitutes in foods and beverages. The market for such products has grown immensely over time. There are human studies which report their negative effects on glucose metabolism with various results of disturbances in glucose metabolism, weight gain etc. No studies to the best of our knowledge have directly addressed the impact of the NCS on muscle glucose uptake. Muscle tissue can account for over 70 percent of whole-body glucose uptake. Therefore, we examined directly the effect of NCS on muscle cell glucose uptake. We find that aspartame moderately increased insulin stimulated glucose uptake by muscle cells in vitro. But sucralose, saccharin and stevia suppressed insulin stimulated glucose uptake. Sucralose is one of the most often used sweetener in foods and beverages globally it is important to understand its effects on glucose metabolism. Therefore, we explored the mechanism of its inhibition of glucose uptake by using an anti-diabetic nutraceutical which is known to target insulin mediated glucose uptake and metabolism pathways. We show here that the nutraceutical is able to relieve the suppression by sucrose in muscle uptake by a novel mechanism of action. We propose that such nutraceuticals may be useful to combine with sucralose containing products to offset negative effects of the NCS on glucose metabolism.

pharmacology and toxicology↗

bioRxiv2022

A molecular atlas of the human postmenopausal fallopian tube and ovary from single-cell RNA and ATAC sequencing

As part of the Human Cell Atlas initiative, we generated transcriptomic (scRNA-seq; 86,708 cells) and regulatory (scATAC-seq; 59,118 cells) profiles of the normal postmenopausal ovary and fallopian tube (FT) at single-cell resolution. In the FT, 22 cell clusters integrated into 11 cell types, including ciliated and secretory epithelial cells, while the ovary had 17 distinct cell clusters defining 6 major cell types. The dominant cell type in both the postmenopausal ovary and FT was stromal cells, which expressed several genes associated with aging. The fimbrial end of the FT had a significant number and variety of immune cells and active communication with the ovary; the ovary contained mostly stromal cells but few immune cells. The epithelial cells of the normal FT expressed multiple ovarian cancer risk-associated genes (CCDC170, RND3, TACC2, STK33, and ADGB). By integrating paired single-cell transcriptomics and chromatin accessibility data we found that the regulatory landscape of the fimbriae was markedly different from the isthmus and ampulla. Intriguingly, several cell types in the FT had comparable gene expression but different transcriptional regulations. Our single-cell transcriptional and regulatory maps allowed us to disentangle the complex cellular makeup of the postmenopausal FT and ovary and will contribute to a better understanding of gynecologic diseases in menopause.

genomics↗

bioRxiv2022

Nutraceutical supplement targeting multiple molecular steps synergistically enhances muscle glucose uptake and improves in vivo oral glucose disposal

Pre diabetes and type 2 diabetes are increasingly becoming rampant world wide. While there are medications to control blood glucose in type 2 diabetics, currently there are no interventions prescribed for pre diabetes. Alternate strategies to control blood glucose are needed either to act alone in pre diabetes or as supplement to the existing drugs for type 2 diabetes. We report here the targeting of critical molecular steps in muscle glucose uptake and metabolism to result in glucose lowering using a combination of safe vitamins. Our in vitro and in vivo data support the potential for using such vitamin combination for glucose control in pre diabetes and as a supplement in type 2 diabetics.

pharmacology and toxicology↗

bioRxiv2022

Immunomodulatory effect of mycobacterial outer membrane vesicles coated nanoparticles

Tuberculosis (TB) is one of the most widely prevalent infectious diseases that cause significant mortality. Bacillus Calmette-Guerin (BCG), the current TB vaccine used in clinics, shows variable efficacy and has safety concerns for immunocompromised patients. There is a need to develop new and more effective TB vaccines. Outer membrane vesicles (OMVs) are vesicles released by Mycobacteria that contain several lipids and membrane proteins and act as a good source of antigens to prime immune response. However, the use of OMVs as vaccines has been hampered by their heterogeneous size and low stability. Here we report that mycobacterial OMVs can be stabilized by coating over uniform-sized 50 nm gold nanoparticles. The OMV-coated gold nanoparticles (OMV-AuNP) show enhanced uptake and activation of macrophages and dendritic cells. Proteinase K and TLR inhibitor studies demonstrated that the enhanced activation was attributed to proteins present on OMVs and was mediated primarily by TLR2 and TLR4. Mass spectrometry analysis revealed several potential membrane proteins that were common in both free OMVs and OMV-AuNP. Such strategies may open up new avenues and the utilization of novel antigens for developing TB vaccines.

bioengineering↗