Biology subjects

Natarajan, M.

Publications and source records attributed to Natarajan, M..

7 recordsLinked to original sources

bioRxiv2025

Binding Kinetics, Bias, Receptor Internalization and Effects on Insulin Secretion in vitro and in vivo of a Novel GLP-1R/GIPR Dual Agonist, HISHS-2001

The use of incretin analogues has emerged in recent years as an effective approach to achieve both enhanced insulin secretion and weight loss in type 2 diabetes (T2D) patients. Agonists which bind and stimulate multiple receptors have shown particular promise. However, off target effects, including nausea and diarrhoea, remain a complication of using these agents, and modified versions with optimized pharmacological profiles and/or biased signaling at the cognate receptors are increasingly sought. Here, we describe the synthesis and properties of a molecule which binds to both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (GLP-1R and GIPR) to enhance insulin secretion. HISHS-2001 shows increased affinity at the GLP-1R, as well as a tendency towards reduced internalization and recycling at this receptor versus FDA-approved dual GLP-1R/GIPR agonist tirzepatide. HISHS-2001 also displayed significantly greater bias towards cAMP generation versus {beta}-arrestin 2 recruitment compared to tirzepatide. In contrast, Gs recruitment was lower versus tirzepatide at the GLP-1R, but higher at the GIPR. Administered to obese hyperglycaemic db/db mice, HISHS-2001 increased circulating insulin whilst lowering body weight and HbA1c with similar efficacy to tirzepatide at substantially lower doses. Thus, HISHS-2001 represents a novel dual receptor agonist with an improved pharmacological profile.

pharmacology and toxicology↗

bioRxiv2022

Machine-learning based detection of adventitious microbes in T-cell therapy cultures using long read sequencing

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP<71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimised experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 CFU / mL using metagenomic classification. Machine learning can be coupled with long read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP<71> organisms to 10 CFU / mL. ImportanceThis research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long read Nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

bioinformatics↗

bioRxiv2022

Lola-I is a developmentally regulated promoter pioneer factor

While enhancers are often regulated at the level of accessibility by pioneer factors, promoters tend to be constitutively accessible and poised for activation by paused Pol II -- thus are often not considered as sites of developmental regulation. Here we show that the accessibility of promoters and the acquisition of paused Pol II can be subject to developmental regulation by pioneer factors. We show that Lola-I, a Drosophila zinc finger transcription factor, is ubiquitously expressed at the end of embryogenesis and causes its target promoters to become accessible and acquire paused Pol II throughout the embryo. This promoter transition is required but not sufficient for tissue-specific target gene expression. Lola-I mediates this function by binding to the edges of the promoter nucleosomes, which leads to their depletion, similar to the action of pioneer factors at enhancers. These results uncover a level of regulation for promoters that is normally found at enhancers, providing further evidence that promoters and enhancers display unexpectedly similar characteristics.

genomics↗

bioRxiv2020

TATA and paused promoters active in differentiated tissues have distinct expression characteristics

Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this difference affects their tissue-specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active at all stages of development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late-stage Drosophila embryos to analyze the properties of promoter types. Using tissue-specific Pol II ChIP-seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. This difference between promoter types is associated with different chromatin accessibility in ATAC-seq data and different expression characteristics in single-cell RNA data. The results suggest that promoter types have optimized different promoter properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue-specific effector genes have evolved to use two different strategies for their differential expression across tissues.

genomics↗

bioRxiv2019

An atlas of transcription factors expressed in the Drosophila melanogaster pupal terminalia

During development, transcription factors and signaling molecules govern gene regulatory networks to direct the formation of unique morphologies. As changes in gene regulatory networks are often implicated in morphological evolution, mapping transcription factor landscapes is important, especially in tissues that undergo rapid evolutionary change. The terminalia (genital and anal structures) of Drosophila melanogaster and its close relatives exhibit dramatic changes in morphology between species. While previous studies have found network components important for patterning the larval genital disc, the networks governing adult structures during pupal development have remained uncharted. Here, we performed RNA-seq in whole Drosophila melanogaster terminalia followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the terminalia is highly patterned during pupal stages and that specific transcription factors mark separate structures and substructures. Our results are housed online in a searchable database (flyterminalia.pitt.edu) where they can serve as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.\n\nSummaryWe performed RNA-seq in whole Drosophila melanogaster terminalia (genitalia and analia) followed by in situ hybridization for 100 highly expressed transcription factors during pupal development. We find that the pupal terminalia is highly patterned with specific transcription factors marking separate structures and substructures. Our results are housed online in a searchable database (flyterminalia.pitt.edu) where they can serve as a resource for the community. This work lays a foundation for future investigations into the gene regulatory networks governing the development and evolution of Drosophila terminalia.

genetics↗

bioRxiv2017

Force dependence of filopodia adhesion: involvement of myosin II and formins

Filopodia are dynamic membrane protrusions driven by polymerization of an actin filament core, mediated by formin molecules at the filopodia tips. Filopodia can adhere to the extracellular matrix and experience both external and cell generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we induced sustained growth of filopodia by applying pulling force to their tips via attached fibronectin-coated beads trapped by optical tweezers. Strikingly, pharmacological inhibition or knockdown of myosin IIA, which localized to the base of filopodia, resulted in weakening of filopodia adherence strength. Inhibition of formins, which caused detachment of actin filaments from formin molecules, produced similar effect. Thus, myosin IIA-generated centripetal force transmitted to the filopodia tips through interactions between formins and actin filaments are required for filopodia adhesion. Force-dependent adhesion led to preferential attachment of filopodia to rigid versus fluid substrates, which may underlie cell orientation and polarization.

cell biology↗

bioRxiv2017

Microtubule regulation of integrin-based adhesions is mediated by myosin-IIA

The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesions, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin-IIA filaments. Myosin-IIA filaments, augmenting the focal adhesions and disrupting the podosomes, are indispensable effectors in the microtubule-dependent regulation of integrin-mediated adhesions. Myosin-IIA filament assembly depends on Rho activation by the RhoGEF, GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capturing by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the myosin-IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.

cell biology↗