Mutations in a Guanylate Cyclase GCY-35/GCY-36 Modify Bardet-Biedl Syndrome–Associated Phenotypes in Caenorhabditis elegans

Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants. Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C. elegans bbs mutants, may also contribute to some BBS features in other organisms

Utilizing a Microbial Natural Product to Investigate Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis

Fungal pathogens cause life-threatening infections in immunocompetent and immunocompromised individuals. The three most prevalent fungal pathogens are Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. There is a limited repertoire of antifungals in clinical use, with treatment of invasive infections restricted to polyenes, azoles, and echinocandins. The emergence of drug resistance now threatens the utility of antifungals, necessitating the development of novel strategies to block the emergence of drug resistance and restore antifungal activity against resistant pathogens. My research explores the mechanisms by which the natural product beauvericin enhances azole efficacy, blocks the emergence of azole resistance, inhibits virulence traits, and renders resistant pathogens responsive to treatment in mammalian infection models. My results demonstrate that beauvericin increases azole activity against Saccharomyces cerevisiae and pathogenic fungi such as C. albicans, C. neoformans, and A. fumigatus. Harnessing genome sequencing of S. cerevisiae beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Genome sequence analysis of beauvericin-resistant mutants in C. albicans reveals mutations in genes encoding the transcription factors Zcf29 and Tac1. Transcriptional profiling and chromatin immunoprecipitation coupled to microarray analyses reveals that Zcf29 binds to and regulates the expression of multidrug transporter genes. Beyond drug resistance, I also discovered that beauvericin blocks the C. albicans morphogenetic transition from yeast to filamentous growth, a key virulence trait, in response to diverse host-relevant cues. Thus, my work establishes that beauvericin abrogates antifungal drug resistance and blocks C. albicans morphogenesis, providing a powerful strategy for the development of combination therapy to treat life-threatening fungal infections.Ph.D

HUMAN CONNECTOME PROJECT (HCP)

This project deals with the nervous system and its function in brain. Here connectome means the microscopic neural connectivity and its mapping between all the neurons present in the brain which further represents their graphical representation on the visual screen also which will further help us to zoom into a region to explore the cells and the functions depending on it and taking this one step ahead the memory implementation in human brain so it will be used as a memory unit except the fact that it will not effect the brain’s existing memory so a person can live his/her daily life too though the basic step is to implement this idea in real life is to study and understand about functioning and working of brain and by activating maximum and achievable amount of inactive brain cells though this step could be revolutionary. Here the research is on the ancient Indian(Vedic), and some new techniques which also includes some today’s technology i.e.; brainstorming, BCI(machine which helps to locate brain in a graphical manner on a visual screen), and etc. , of boosting up our some inactive brain part. The central problem on which this research is written, is the inactive brain cells between the teen age to old age. The basic design of the study is to implement our old Indian techniques and some others to achieve the brain’s usage limit and to figure out the way to exceed it too which will further help us to store data digitally in brain. Nowadays brainstorming is a trend to activate the inactive brain cells which opens energy centers in our body which include some extraordinary activities that is almost impossible to do by any normal human being but researchers have found that Indian Yoga is way more powerful than the brainstorming activities in a manner to be body fit too and so far we found that this the only way to increase brain capacity and its usage though in future it might be possible due to some new high-tech machines

Design and synthesis of fungal-selective resorcylate aminopyrazole Hsp90 inhibitors

The molecular chaperone Hsp90, essential in all eukaryotes, plays a multifaceted role in promoting survival, virulence, and drug resistance across diverse pathogenic fungal species. The chaperone is also critically important, however, to the pathogen's human host, preventing the use of known clinical Hsp90 inhibitors in antifungal applications due to concomitant host toxicity issues. With the goal of developing Hsp90 inhibitors with acceptable therapeutic indices for the treatment of invasive fungal infections, we initiated a program to design and synthesize potent inhibitors with selective activity against fungal Hsp90 isoforms over their human counterparts. Building on our previously reported derivatization of resorcylate natural products to produce fungal-selective compounds, we have developed a series of synthetic aminopyrazole-substituted resorcylate amides with broad, potent, and fungal-selective Hsp90 inhibitory activity. Herein we describe the synthesis of this series, as well as biochemical structure-activity relationships driving selectivity for the Hsp90 isoforms expressed by Cryptococcus neoformans and Candida albicans, two pathogenic fungi of major clinical importance.R01 AI120958 - NIAID NIH HHSAccepted manuscript2021-03-1

Structural basis for species-selective targeting of Hsp90 in a pathogenic fungus

New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy

Dual action antifungal small molecule modulates multidrug efflux and TOR signaling

There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, we establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders antifungal-resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating the protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for treating fungal infectious disease that evades resistance