Biology subjects

Kulkarni, M.

Publications and source records attributed to Kulkarni, M..

29 records · Page 2Linked to original sources

bioRxiv2023

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4 dicarboxylate transporter

The COVID-19 pandemic has hastened the problem of nosocomial drug-resistant pathogens and is exerting a huge toll on hospitalized patients in critical care settings. Most small molecule antibiotics are susceptible to bacterial resistance mechanisms and constantly becoming ineffective leading to rapid shrinkage of the antibiotic armamentarium. Pseudomonas aeruginosa, one of the most common pathogens in hospital-born infection and considered critically dangerous by WHO and CDC, is extremely difficult to treat with frontline drugs, the carbapenems. In this study, we developed a camelid antibody fragment (VHH) library against whole P. aeruginosa and isolated a highly potent neutralizing hit (PsC23) that selectively targets P. aeruginosa . At 25 {micro}g/mL, PsC23 inhibited growth of the ATCC 27853 and a locally isolated carbapenem resistant MCC 50428 P. aeruginosa. The target of PsC23 is the C4 dicarboxylate transporter that transports C4 metabolites to the glyoxylate shunt during oxidative stress that is present in pathogens but not the human host. This ultimately results in the blockade of the shunt affecting bacterial energy transduction that leads to disruption of drug efflux. Interestingly, in a neutropenic mouse with MCC 50428 systemic infection, PsC23 in combination with meropenem completely reversed the drug resistance and eliminated the pathogens from the blood. PsC23 was stable in human serum and had no hemolytic or cytotoxic effect on human cells. Taken together, this VHH if co administered with the currently available carbapenems would reverse carbapenem resistance and could be used to effectively control P. aeruginosa in critical care settings.

molecular biology↗

bioRxiv2022

Comparative Analysis of RNA Secondary Structure Accuracy on Predicted RNA 3D Models

RNA structure is conformationally dynamic, and accurate all-atom tertiary (3D) structure modeling of RNA remains challenging with the prevailing tools. Secondary structure (2D) information is the standard prerequisite for most RNA 3D modeling. Despite several 2D and 3D structure prediction tools proposed in recent years, one of the challenges is to choose the best combination for accurate RNA 3D structure prediction. Here, we benchmarked seven small RNA PDB structures (40 to 90 nucleotides) with different topologies to understand the effects of different 2D structure predictions on the accuracy of 3D modeling. The current study explores the blind challenge of 2D to 3D conversions and highlights the performances of de novo RNA 3D modeling from their predicted 2D structure constraints. Our results show that conformational sampling-based methods such as SimRNA and IsRNA1 depend less on 2D accuracy, whereas motif-based methods account for 2D evidence. Our observations illustrate the disparities in available 3D and 2D prediction methods and may further offer insights into developing topology-specific or family-specific RNA structure prediction pipelines.

bioinformatics↗

bioRxiv2022

Spatiotemporal mapping of immune and stem cell dysregulation after volumetric muscle loss

Volumetric muscle loss (VML) is an acute trauma that results in persistent inflammation, supplantation of muscle tissue with fibrotic scarring, and decreased muscle function. The cell types, nature of cellular communication and tissue locations that drive the aberrant VML response have remained elusive. Herein, we used spatial transcriptomics integrated with single-cell RNA sequencing on mouse and canine models administered VML. We observed VML engenders a unique spatial pro-fibrotic pattern driven by crosstalk between macrophages and fibro-adipogenic progenitors that was conserved between murine and canine models albeit with varying kinetics. This program was observed to restrict muscle stem cell mediated repair and targeting this circuit in a murine model resulted in increased regeneration and reductions in inflammation and fibrosis. Collectively, these results enhance our understanding of the immune cell-progenitor cell-stem cell crosstalk that drives regenerative dysfunction and provides further insight into possible avenues for fibrotic therapy exploration.

cell biology↗

bioRxiv2022

Design of a systemic small molecule clinical STING agonist using physics-based simulations and artificial intelligence

The protein STING (stimulator of interferon genes) is a central regulator of the innate immune system and plays an important role in antitumor immunity by inducing the production of cytokines such as type I interferon (IFN). Activation of STING stems from the selective recognition of endogenous cyclic dinucleotides (CDNs) by the large, polar, and flexible binding site, thus posing challenges to the design of small molecule agonists with drug-like physicochemical properties. In this work we present the design of SNX281, a small molecule STING agonist that functions through a unique self-dimerizing mechanism in the STING binding site, where the ligand dimer approximates the size and shape of a cyclic dinucleotide while maintaining drug-like small molecule properties. SNX281 exhibits systemic exposure, STING-mediated cytokine release, strong induction of type I IFN, potent in vivo antitumor activity, durable immune memory, and single-dose tumor elimination in mouse models via a Cmax-driven pharmacologic response. Bespoke computational methods - a combination of quantum mechanics, molecular dynamics, binding free energy simulations, and artificial intelligence - were developed during the course of the project to design SNX281 by explicitly accounting for the unique self-dimerization mechanism and the large-scale conformational change of the STING protein upon activation. Over the course of the project, we explored millions of virtual molecules while synthesizing and testing only 208 molecules in the lab. This work highlights the value of a multifaceted computationally-driven approach anchored by methods tailored to address target-specific problems encountered along the project progression from initial hit to the clinic.

cancer biology↗

bioRxiv2022

Human and murine Cryptococcus neoformans infection selects for common genomic changes in an environmental isolate.

A pet cockatoo was the suspected source of Cryptococcus neoformans recovered from the cerebral spinal fluid (CSF) of an immunocompromised patient with cryptococcosis based on the molecular analyses available in 2000. Here we report whole genome sequence analysis of the clinical and cockatoo strains. Both are closely related MAT strains belonging to the VNII lineage, confirming that the human infection likely originated from pet bird exposure. The two strains differ by 61 single nucleotide polymorphisms, including 8 nonsynonymous changes involving 7 genes. To ascertain whether changes in these genes are selected during mammalian infection, we passaged the cockatoo strain in mice. Remarkably, isolates obtained from mouse tissue possess a frame-shift mutation in one of the seven genes altered in the human sample, a gene predicted to encode a SWI-SNF chromatin-remodeling complex protein. Both cockatoo and patient strains as well as mouse passaged isolates obtained from brain tissue had a premature stop codon in a homolog of ZFC3, a predicted single-zinc finger containing protein, which is associated with larger capsules when deleted and appears to have reverted to a full-length protein in the mouse passaged isolates obtained from lung tissue. The patient strain and mouse passaged isolates show variability in the expression of virulence factors, with differences in capsule size, melanization, and rates on non-lytic expulsion from macrophages observed. Our results establish that environmental strains undergo genomic and phenotypic changes during mammalian passage, suggesting that animal virulence can be a mechanism for genetic change and that the genomes of clinical isolates may provide a readout of mutations acquired during infection.

genomics↗

bioRxiv2022

Melanization is an important antifungal defense mechanism in Galleria mellonella hosts

A key component of insect immunity is melanin encapsulation of microbes. Melanization is also a part of an immune process known as nodulation, which occurs when insect hemocytes surround microbes and produce melanin. Insect nodules are analogous to mammalian immune granulomas. Melanin is believed to kill microbes through the production of toxic intermediates and oxidative damage. However, it is unclear to what extent immune melanin is directly fungicidal during infections of insect hosts. We reported previously that C. neoformans cells are encapsulated with host-derived melanin within hemocyte nodules. Here we report an association between melanin-based immune responses by Galleria mellonella wax moth larvae and fungal cell death of C. neoformans during infection. To monitor melanization in situ, we applied a tissue-clearing technique to G. mellonella larvae, revealing that nodulation occurs throughout the organism. Further, we developed a protocol for time-lapse microscopy of extracted hemolymph following exposure to fungal cells, which allowed us to visualize and quantify the kinetics of the melanin-based immune response. Using this technique, we found evidence that cryptococcal melanins and laccase enhance immune melanization in hemolymph. We used these techniques to also study the fungal pathogen Candida albicans infections of G. mellonella. We find that the yeast form of C. albicans was the primary targets of host melanization, while filamentous structures were melanin-evasive. Approximately 23% of melanin-encapsulated C. albicans yeast survive and break through the encapsulation. Overall, our results provide direct evidence that the melanization reaction functions as a direct antifungal mechanism in insect hosts.

microbiology↗

bioRxiv2021

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

Unicellular eukaryotes are suggested to undergo self-inflicted destruction. However, molecular details are sparse by comparison to the mechanisms of cell death known for human cells and animal models. Here we report a molecular pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization and cell death. Following exposure to heat-ramp conditions, a model of environmental stress, we observed that yeast cell death occurs over several hours, suggesting an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 adaptor complex. AP-3 promotes stress-induced cell death through its Arf1-GTPase-dependent vesicle trafficking function, which is required to transport and install proteins on the vacuole/lysosome membrane, including a death-promoting protein kinase Yck3. Time-lapse microscopy revealed a sequence of events where AP-3-dependent vacuole permeability occurs hours before the loss of plasma membrane integrity. An AP-3-dependent cell death pathway appears to be conserved in the human pathogen Cryptococcus neoformans.

cell biology↗

bioRxiv2021

Expression of extracellular Hsp90 is a molecular signature of T cell activation, providing a means to image and target T Cell activation in autoimmune disease

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified an extracellularly expressed form of Hsp90, eHsp90, that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered eHsp90 inhibitor, to quantify the effect of T cell activation on the expression of eHsp90 in human and mouse T cells. In cell based assays, stimulation of human T cells induced a 20-fold increase in eHsp90 expression at the plasma membrane, suggesting trafficking of eHsp90 is acutely regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in delaying the onset and progression of disease in the arthritis model. Our results suggest eHsp90 expression at the plasma membrane of T cells is a molecular marker of autoimmune induced activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis and inflammatory bowel disease. PerspectiveT cells mediate many maladaptive disease pathologies such as autoimmune disorders. Many immunosuppressants in clinical use act by preventing the activation of T cells. However, this approach can lead to increased rates of opportunistic infection and malignancy. Here, we show for the first time that eHsp90 is upregulated during T cell activation in response to exogenous ligands. Therefore, pharmacological inhibition of eHsp90 may provide a means to target only activated T cells for immunosuppression. Selective targeting of only activated T cells could be efficacious against autoimmune disease without increasing the incidence of undesirable outcomes. Additionally, the presence of eHsp90 on the surface of activated T cells could be diagnostically useful to track the status and progression of autoimmune disease in patients.

pharmacology and toxicology↗

bioRxiv2021

Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180{degrees} around the C{beta} - C{gamma} axis ({chi}2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that{chi} 2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed{chi} 1 (N - C) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

biophysics↗

bioRxiv2020

PTPN11/SHP2 negatively regulates growth in breast epithelial cells: implications on tumorigenesis

PTPN11/SHP2, a non-receptor protein tyrosine phosphatase is a prominent target of the receptor tyrosine kinase that participates in positive feedback signalling of the human epidermal growth factor receptors and helps in growth and migration. PTPN11/SHP2 is widely believed to be an oncoprotein, although its possible tumor-suppressor role is also reported. Our analysis of breast cancer metadata shows, PTPN11/SHP2 copy number loss in luminal A subtype is correlated to poor disease-specific survival and late-stage cancer at diagnosis. Analysis of the level 4 Reverse Phase Protein Array (RPPA) data available on the TCGA database resulted in positive correlations between the lower expression levels of constitutively active variant, the phospho-SHP2-Y542, of PTPN11/SHP2 and larger tumor size and lymph node positivity. We experimentally examined possible negative regulation of growth by PTPN11/SHP2 using MCF10A, a normal breast epithelial cell line. Knock-down of PTPN11/SHP2 resulted in increased cell migration, cell shape changes to mesenchymal morphology, and increased survival in cells treated with epirubicin, a DNA-damaging drug. However, it did not alter the rate of cell proliferation. It is possible that PTPN11/SHP2 might function as a tumor suppressor by potentiating proliferating cells with increased cell migration and resistance to apoptosis. Statement of SignificanceMolecules like PTPN11/SHP2, among many others that show dual specificity in tumorigenesis in the same tissue depending on the upstream signaling cues, present challenges in the field of targeted drug therapy. This study puts forth the importance of understanding the mechanism of one of the two outcomes and thereby helps better clinical management of a subgroup of cancer.

cancer biology↗

bioRxiv2018

Overwhelming presence of non-food vegetation undermines the reliability of NDVI as a surrogate of forage abundance for a large herbivore in a tropical forest habitat

The use of remotely-sensed vegetation indices has increased in wildlife studies but field-based support for their utility as a measure of forage availability largely comes from open-canopy habitats. In this paper, we assessed whether the popular vegetation index, NDVI, actually represents forage availability for Asian elephants in a southern Indian tropical forest. We found that the number of food species was a small percentage of all plant species, and the abundance of food species compared to total species abundance varied across different vegetation categories. NDVI was not a good measure of food abundance in any vegetation category partly because of a) small to moderate proportional abundances of food species relative to the total abundance of all species in that category (herb and shrub categories), b) abundant overstorey vegetation resulting in low correlations between NDVI and food abundance despite a high proportional abundance of food species and a concordance between total abundance and food species abundance (graminoid category), and c) the relevant variables measured and important as food at the ground level (count and GBH) not being related to primary productivity (trees and recruits). NDVI had a negative relationship with the total abundance of graminoids, which represent a bulk of elephant and other herbivore diet, because of the presence of other vegetation types and canopy cover that positively explained NDVI. We also found that spatially interpolated total graminoid abundance modelled from field data outperformed NDVI in predicting total graminoid abundance, although interpolation models of food graminoid species abundance were not satisfactory. Our results reject the utility of NDVI as a surrogate of elephant forage abundance in tropical forests with multistorey vegetation, a finding that has implications for studies of other herbivores in such habitats.

ecology↗