Genetically Encoded Tools for The Discovery and Biosynthesis of Bioactive Small Molecules in Escherichia Coli

The identification of molecules that bind to and modulate disease-relevant proteins is a central challenge in medicine. Traditionally, these molecules are discovered through laborious screens of large compound libraries; in contrast, nature is constantly identifying biologically active molecules through evolutionary pressures on genes producing natural products. Inspired by evolution, engineered selection pressures have been developed in biological systems like Escherichia coli to identify proteins with new or improved activities. Similar strategies for identifying small molecules based on their activity are lacking, but could, combined with existing tools for producing natural products in microbes, provide an efficient method for molecular discovery. Chapter 1 reviews the role of natural products (particularly, terpenoids) in medicine, traditional paradigms for natural product drug discovery, and biological tools to supplement these paradigms. In Chapter 2, I develop a genetically encoded system to detect the activity of an important drug target&mdash;protein tyrosine phosphatase 1B (PTP1B) in E. coli. This system identified three terpenoid inhibitors, including two with surprising selectivity against other phosphatases. In Chapter 3, I use the same system to evolve a terpene synthase towards production of a PTP1B inhibitor, discovering mutants with higher production of and/or narrowed product profiles. Finally, in Chapter 4, I modify the system to respond to the activity of the viral proteases HIV1-protease and 3-chymotrypsin-like protease from SARSCoV2. The results provide a framework for building and characterizing other protease-based systems. Together, these three studies (i) provide new genetically encoded tools for detecting the activity of a drug target in a microbe and (ii) demonstrate how these tools can facilitate screening for biologically active molecules in microbes.</p

AstroSat observation of rapid Type-I thermonuclear burst from the low mass X-ray binary GX 3+1

We report the results of an observation of low mass X-ray binary GX 3+1 with {\it AstroSat}'s Large Area X-ray Proportional Counter (LAXPC) and Soft X-ray Telescope (SXT) instruments on-board for the first time. We have detected one Type-1 thermonuclear burst ($\sim$ 15 s) present in the LAXPC 20 light curve, with a double peak feature at higher energies and our study of the hardness-intensity diagram reveals that the source was in a soft banana state. The pre-burst emission could be described well by a thermally Comptonised model component. The burst spectra is modelled adopting a time-resolved spectroscopic method using a single color blackbody model added to the pre-burst model, to monitor the parametric changes as the burst decays. Based on our time-resolved spectroscopy, we claim that the detected burst is a photospheric radius expansion (PRE) burst. During the PRE phase, the blackbody flux is found to be approximately constant at an averaged value $\sim$ 2.56 in $10^{-8}$ ergs s$^{-1}$ cm$^{-2}$ units. On the basis of literature survey, we infer that \textit{AstroSat}/LAXPC 20 has detected a burst from GX 3+1 after more than a decade which is also a PRE one. Utilising the burst parameters obtained, we provide a new estimation to the source distance, which is $\sim$ 9.3 $\pm$ 0.4 kpc, calculated for an isotropic burst emission. Finally, we discuss and compare our findings with the published literature reports.Comment: 14 pages, 10 figures, accepted for publication in The Journal of Astrophysics and Astronom

Submicrometer Pattern Fabrication by Intensification of Instability in Ultrathin Polymer Films under a Water-Solvent Mix

Dewetting of ultrathin (< 100 nm) polymer films, by heating above the glass transition, produces droplets of sizes of the order of microns and mean separations between droplets of the order of tens of microns. These relatively large length scales are because of the weak destabilizing van der Waals forces and the high surface energy penalty required for deformations on small scales. We show a simple, one-step versatile method to fabricate sub-micron (>~100 nm) droplets and their ordered arrays by room temperature dewetting of ultrathin polystyrene (PS) films by minimizing these limitations. This is achieved by controlled room temperature dewetting under an optimal mixture of water, acetone and methyl-ethyl ketone (MEK). Diffusion of organic solvents in the film greatly reduces its glass transition temperature and the interfacial tension, but enhances the destabilizing field by introduction of electrostatic force. The latter is reflected in a change in the exponent, n of the instability length scale, {\lambda} ~h^n, where h is the film thickness and n = 1.51 \pm 0.06 in the case of water-solvent mix, as opposed to its value of 2.19 \pm 0.07 for dewetting in air. The net outcome is more than one order of magnitude reduction in the droplet size as well as their mean separation and also a much faster dynamics of dewetting. We also demonstrate the use of this technique for controlled dewetting on topographically patterned substrates with submicrometer features where dewetting in air is either arrested, incomplete or unable to produce ordered patterns

Multi-messenger searches via IceCube’s high-energy neutrinos and gravitational-wave detections of LIGO/Virgo

We summarize initial results for high-energy neutrino counterpart searches coinciding with gravitational-wave events in LIGO/Virgo\u27s GWTC-2 catalog using IceCube\u27s neutrino triggers. We did not find any statistically significant high-energy neutrino counterpart and derived upper limits on the time-integrated neutrino emission on Earth as well as the isotropic equivalent energy emitted in high-energy neutrinos for each event