Biology subjects

Kamat, N.

Publications and source records attributed to Kamat, N..

12 recordsLinked to original sources

bioRxiv2024

A synthetic method to assay polycystin ion channel biophysics

Ion channels are biological transistors that control ionic flux across cell membranes to regulate electrical transmission and signal transduction. They are found in all biological membranes and their conductive state kinetics are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident ionophores. Here we describe a fully synthetic method to assay functional properties of polycystin channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their oligomeric assembly, membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate channels resistant to biophysical analysis and pharmacological characterization.

synthetic biology↗

bioRxiv2023

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

bioengineering↗

bioRxiv2022

Engineering transmembrane signal transduction in synthetic membranes using two-component systems

Cells use signal transduction across their membranes to sense and respond to a wide array of chemical and physical signals. Creating synthetic systems which can harness cellular signaling modalities promises to provide a powerful platform for biosensing and therapeutic applications. As a first step towards this goal, we investigated how bacterial two-component systems can be leveraged to enable transmembrane-signaling with synthetic membranes. Specifically, we demonstrate that a bacterial two-component nitrate-sensing system (NarX-NarL) can be reproduced outside of a cell using synthetic membranes and cell-free protein expression systems. We find that performance and sensitivity of the two-component system can be tuned by altering the biophysical properties of the membrane in which the histidine kinase (NarX) is integrated. Through protein engineering efforts, we modify the sensing domain of NarX to generate sensors capable of detecting an array of ligands. Finally, we demonstrate that these systems can sense ligands in relevant sample environments. By leveraging membrane and protein design, this work helps reveal how transmembrane sensing can be recapitulated outside of the cell, adding to the arsenal of deployable cell-free systems primed for real world biosensing. Significance StatementCells detect and respond to environmental and chemical information by using a combination of membrane proteins and genetic polymers. Recapitulation of this behavior in synthetic systems holds promise for engineering biosensors and therapeutics. Using the nitrate-sensing bacterial two-component system as a model, we demonstrate methods to reproduce and tune transmembrane signaling in synthetic lipid membranes, leading to the synthesis of genetically programmed proteins. Through this study, we gain insight into how membrane augmented cell-free systems can be used as a platform to characterize membrane-receptor interactions and engineer new biosensors.

synthetic biology↗

bioRxiv2022

Enhancing functionalized liposome avidity to cells via lipid phase separation

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickelnitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.

bioengineering↗

bioRxiv2022

Probing the force-from-lipid mechanism with synthetic polymers

A central feature of mechanotransduction is the ability of mechanosensitive channels to respond to mechanical stimuli from the surrounding lipid bilayer. Accordingly, the mechanical properties of membranes should play an important role in modulating force transmission to embedded channels, yet the nature of this relationship remains unclear for a wide class of mechanosensitive channels across prokaryotic and eukaryotic systems. Here, we use a synthetic amphiphile to modulate the membrane mechanical properties of cell-derived vesicles and probe channel activation. Using precise membrane mechanical characterization approaches that have rarely been used in conjunction with electrophysiology techniques, we directly characterize three membrane properties and the activation threshold of the E. coli mechanosensitive channel of large conductance (MscL). Our study reveals that decreases in the membrane area expansion modulus, KA, and bending rigidity, kc, correlate with increases in the pressure required to activate MscL and that this effect is reproducible with the mammalian channel, TREK-1. MD simulations demonstrate that polymer-mediated changes in interfacial tension is the best mechanism to describe these experimental results. Together, our results bolster the force-from-lipids mechanism by demonstrating the generality of the relationship between changes in specific membrane mechanical properties and the gating pressure of MscL and TREK-1. In addition, our results reveal the mechanical mechanism by which membrane amphiphiles alter the activity and sensitivity of mechanosensitive channels through changes in long-range force transmission.

biophysics↗

bioRxiv2022

Enzymatically-active bacterial microcompartments follow substrate gradients and are protected from aggregation in a cell-free system

The ability to dynamically control organelle movement and position is essential for cellular function. Yet the underlying mechanisms driving this organization have not been fully resolved. Here, we draw from recent experimental observations and theoretical models of enzyme chemotaxis to demonstrate the chemotaxis of a bacterial organelle, the 1,2 propanediol (1,2-PD) utilization bacterial microcompartment (MCP) from Salmonella enterica. Upon encapsulating MCPs in a cell-like, biomimetic compartment, we observed the directed movement of MCPs along an external gradient of substrate. Our analysis shows that MCPs not only chemotax towards their substrate but also that enzymatic activity and substrate turnover protect them against large-scale aggregation. Our results provide a first experimental demonstration of organelle chemotaxis in a synthetic cellular system and support a recent theoretical model of chemotaxis. Together this work reveals a potentially significant driver of organelle organization while contributing to the construction of synthetic cell-like materials.

synthetic biology↗

bioRxiv2021

Elucidating design principles for engineering cell-derived vesicles to inhibit SARS-CoV-2 infection

The ability of pathogens to develop drug resistance is a global health challenge. The SARS-CoV-2 virus presents an urgent need wherein several variants of concern resist neutralization by monoclonal antibody therapies and vaccine-induced sera. Decoy nanoparticles--cell-mimicking particles that bind and inhibit virions--are an emerging class of therapeutics that may overcome such drug resistance challenges. To date, we lack quantitative understanding as to how design features impact performance of these therapeutics. To address this gap, here we perform a systematic, comparative evaluation of various biologically-derived nanoscale vesicles, which may be particularly well-suited to sustained or repeated administration in the clinic due to low toxicity, and investigate their potential to inhibit multiple classes of model SARS-CoV-2 virions. A key finding is that such particles exhibit potent antiviral efficacy across multiple manufacturing methods, vesicle subclasses, and virus-decoy binding affinities. In addition, these cell-mimicking vesicles effectively inhibit model SARS-CoV-2 variants that evade monoclonal antibodies and recombinant protein-based decoy inhibitors. This study provides a foundation of knowledge that may guide the design of decoy nanoparticle inhibitors for SARS-CoV-2 and other viral infections.

bioengineering↗

bioRxiv2021

Lipid phase separation in vesicles enhances TRAIL-mediated cytotoxicity

Ligand spatial presentation and density play important roles in many signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Currently, Janus particles are most often used to spatially control ligand conjugation, but the technological challenge of manufacturing Janus particles limits adoption for translational applications. Here, we demonstrate that lipid phase separation can be used to spatially control protein presentation onto lipid vesicles. We used this system to study the density dependence of TNF-related apoptosis inducing ligand (TRAIL), a model therapeutic protein that exhibits greater cytotoxicity to cancer cells when conjugated onto a vesicle surface than when administered as a soluble protein. Using assays for apoptosis and caspase activity, we show that phase separated TRAIL vesicles induced higher cytotoxicity to Jurkat cancer cells than uniformly-conjugated TRAIL vesicles, and enhanced cytotoxicity was dependent on the TRAIL domain density. We then assessed this relationship in other cancer cell lines and demonstrated that phase separated TRAIL vesicles only enhanced cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, was unaffected by the TRAIL density. These results indicate unique signaling requirements for each TRAIL receptor and how TRAIL therapy could be tailored depending on the relative levels of expression for cancer receptors of interest. Overall, this work demonstrates a readily adoptable method to control protein conjugation and density on bilayer vesicles that can be easily adopted to other therapeutic nanoparticle systems to improve receptor signaling of nanoparticles targeted to cancer and diseased cells.

bioengineering↗

bioRxiv2017

Calcimycocavitological Studies on Seashells from Beaches of North Goa, India

Calcimycocavitology deals with the study of hollowing out of spaces in hard calcareous seashells by the fungi called calcimycocavites. Endolithic fungi in the shells were first reported and named as the trace fossils in 1889 by Bornet and Flahault. Endolithic fungi bore inside the shell by the process of bioturbation by using organic acids (oxalic acid, citrate) and enzymes (proteases, dehydrogenases and oxidoreductases). Previous reports show the presence of tunnels formed by fungal hyphae in the shells. The present work reports preliminary results of calcimycocavitological studies on the seashells from beaches of north Goa, India. The calcareous sand samples were collected from Arambol, Ashvem, Morjim, Vagator, Anjuna, Baga and Miramar beaches by pool sampling method and were separated into different fractions by using standard sieves. Each fraction of sand was subjected to stereomicroscopic studies which revealed that the sand fraction between 150-250 m included irregular shell fragments showing positive colonization by calcimycocavites. Hydrochloric acid treatment was used to dissolve the shells and release calcimycocavites biomass which was stained with Congo Red and tentatively identified as distinct microfungal forms. SEM studies of the calcareous shell fragments revealed the microtunneling behavior of the fungi. Digital analysis of SEM images using Mountain premium 7.2 software revealed the fine topography of calcimycocavites hyphae along with unidentified presumptive biomineral encrustations. The ecological, biological and biogeochemical implications of the findings are presented with respect to possible role of calcimycocavites in Calcium and Carbon cycling by breakdown of the calcareous shells and release of inorganic and organic components in the ecosystem.

microbiology↗

bioRxiv2017

Fossilized Microbial Forms in Baltic and Goan Amber - A Comparative Pioneer Study

This paper is based on surveys, exploration and standardization of techniques to recover rare amber samples from sands in Goa and identify specimens embedded with distinct microbial fossils based on studies on reference samples of imported Baltic amber. We developed techniques to locate, detect and identify amber samples in local sand. In this pioneer study, we report presumptive microbial forms such as actinobacteria and fungi in amber samples of Goa throwing light on microbial paleobiodiversity. Baltic amber (Succinate) is fossilized resin belonging to the Eocene period (44-49 million years old) derived from the Araucariaceae and Leguminoseae families of trees containing up to 8% of Succinic acid and compounds such as terpenoids and phenolic derivatives. Sooty moulds in the amber fossils have been studied (Schmidt et al., 2014). Samples of imported Baltic amber were validated, studied and used as reference for identification and characterization of amber found in sand of Goa. FTIR Spectroscopic tests diagnostic of presence of Succinate proved that both samples meet the criteria as plant derived Succinate containing products. Fossil fungi in Baltic amber were observed, and compared with similar forms in rare amber fragments of Goa. These samples were subjected to microscopic examination. Actinobacterial and fungal forms embedded in local amber were compared with similar forms found in imported Baltic amber and those published in literature. Detection of fossilized actinobacterial and fungal forms has shown us the potential for further studies for comprehensive collection and microscopic examination of such paleomicrobial forms in rare local amber samples.

evolutionary biology↗

bioRxiv2017

Subsurface Zircons With Presumptive "Biogenic" Inclusions As Potentially Useful Proxies For Studying Precambrian Bygone Biospheres In Goa

This work was inspired by recent report by Bell et al., 2015 who studied potentially biogenic carbon preserved in a 4.1 billion-year-old Zircon and need to assess the potential of Zircons found in Goa. Zircons (ZrSiO4) are naturally occurring silicate minerals which show radioactivity and high ductility and contain traces of Thorium and Uranium useful in Uranium-Thorium /Thorium -230 dating techniques. Zircons can be found in igneous, metamorphic rocks, sedimentary deposits and occurs as a detrital minerals in river and beach sands. Previous reports show that the Zircons can occur in different shapes such as round, elongated and with surface characteristics (Gartner et al.,2013). U-Pb Zircon dating methods had been used to study the continental growth in the western Dharwar craton of southern India (Jayananda et al., 2015). The present study was aimed at detection of subsurface Zircons with biogenic inclusions and assess their use as proxies for studying bygone Precambrium biospheres in Goa. Deep tubewell drilled Cores (60 and 65 m deep from surface) in island of Tiswadi at Taleigao were analyzed by light microscopy, Phase contrast microscopy and SEM to detect and classify the Zircons. In rapid preliminary sampling, total 50 Zircons were identified and 98% indicated the presence of interesting inclusions. These could be bubbles or kerogens or unidentified biological material. Zircons were classified as elongated, slightly rounded with sharp edges and showed widespread variety of surface characteristics like fracturing, cracks, scratches, striations and impact pits which may occur during transport processes. It is suggested that Zircons with presumptive biogenic inclusions can be further studied using techniques such as Raman Spectroscopy, Carbon Isotopic Measurements, X-Ray Microscopy, Trace Element Measurement consistent with Bell et al., 2015. More exhaustive studies have been undertaken to create a detail image database of Zircons from various other local samples to pinpoint those specifically useful for advanced work based on image analysis of the presumptive bioinclusions. Further attempts would be made to develop specific harvesting techniques to select potentially useful Zircons. International collaborations would be sought for applications of advanced techniques to local Zircons. Such studies would shed light on nature of bygone Precambrian biospheres in Goa and help in understanding evolution of life and the impact of plate tectonics and cataclysmic events shaping life on this planet.

evolutionary biology↗

bioRxiv2017

Novel Techniques To Study Ancient Micro Amber From Tropical Beach Sand Reveal A Treasurehouse Of Exceptionally Well Preserved Fossilized Microfungi

Simple, novel techniques developed for separation and simultaneous direct morphometric study of Amber micro fragments (AMF) from tropical beach sand are reported yielding rich information on unidentified fossilized microfungi. Sieves of different mesh sizes were used to separate AMF from tropical beach sand. Fractions below 150 m which proved rich in AMF were used for manual retrieval using stereomicroscope. A handprinted slide microarray having 4 X 12 squares used for microscopic examination of multiple AMF mounts revealed AMF having either rough or smooth surfaces and with or without microinclusions. The microinclusions could be morphologically attributed to fungi. The potential for systematic and comprehensive studies to retrieve and examine AMF at high frequency from tropical beach sand in the world and especially those which are threatened due to sea level rise due to climate change was demonstrated. The potential of retrievable AMF from tropical beach sand in microbiological, metagenomic studies and as biological proxies to reconstruct bygone biospheres has been highlighted.\n\nSummaryNovel techniques for retrieval of AMF and visualization using slide microarray are described. Sand samples from various locations from Goa were collected by pool sampling method. Microscopic study helped to reveal that fraction between 150 and below 53 m contained microscopic fragments of Amber ranging from size of within the size range of 70 m or below and with or without bio inclusions. AMF Specimens with microinclusions such as fungi were identified and studied using standard keys.

evolutionary biology↗