Biology subjects

Singh, Y.

Publications and source records attributed to Singh, Y..

25 records · Page 2Linked to original sources

bioRxiv2020

Overexpression of human alpha-synuclein leads to dysregulated microbiome and metabolites with ageing in a rat model of Parkinson disease

Since Braaks hypothesis stating that sporadic Parkinsons disease follows a specific progression of the pathology from the peripheral to the central nervous system and can be monitored by detecting accumulation of the alpha-Synuclein protein. There is growing interest in understanding how the gut (commensal) microbiome can regulate alpha-Synuclein accumulation which can lead to PD. We studied a transgenic rat model overexpressing the human alpha-Synuclein and found that the protein overexpression resulted in gut alpha-Synuclein expression and aggregation in the gut neurons with advancing age. A progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young transgenic rat model and interestingly this ratio was then increased with aging. This observation was accompanied in older animals by intestinal inflammation, increase gut permeability and a robust alteration in metabolites production characterized by the increase of succinate level in the feces and serum. Manipulation of the gut bacteria by short-term antibiotics treatment revealed a complete loss of short-chain fatty acids (SCFAs) and reduction in succinate levels. Although antibiotics treatment did not change alpha-synuclein expression in the enteric nervous system of the colon, it can reduce alpha-synuclein expression in the olfactory bulb of the transgenic rats. In summary, synchronous with ageing, our data emphasize that the gut microbiome dysbiosis leads to a specific alteration of gut metabolites which are reflected in the serum and can be modulated by the environment.

neuroscience↗

bioRxiv2020

SARS-CoV-2 infection paralyzes cytotoxic and metabolic functions of immune cells

The SARS-CoV-2 virus is the causative agent of the global COVID-19 infectious disease outbreak, which can lead to acute respiratory distress syndrome (ARDS). However, it is still unclear how the virus interferes with immune cell and metabolic functions in the human body. In this study, we investigated the immune response in acute or convalescent COVID19 patients. We characterized the peripheral blood mononuclear cells (PBMCs) using flow cytometry and found that CD8+ T cells were significantly subsided in moderate COVID-19 and convalescent patients. Furthermore, characterization of CD8+ T cells suggested that patients with a mild and moderate course of the COVID-19 disease and convalescent patients have significantly diminished expression of both perforin and granzyme A in CD8+ T cells. Using 1H-NMR spectroscopy, we characterized the metabolic status of their autologous PBMCs. We found that fructose, lactate and taurine levels were elevated in infected (mild and moderate) patients compared with control and convalescent patients. Glucose, glutamate, formate and acetate levels were attenuated in COVID-19 (mild and moderate) patients. In summary, our report suggests that SARS-CoV-2 infection leads to disrupted CD8+ T cytotoxic functions and changes the overall metabolic functions of immune cells.

immunology↗

bioRxiv2020

Comparative Genomic Analysis of Rapidly Evolving SARS CoV-2 Viruses Reveal Mosaic Pattern of Phylogeographical Distribution

The Coronavirus Disease-2019 (COVID-19) that started in Wuhan, China in December 2019 has spread worldwide emerging as a global pandemic. The severe respiratory pneumonia caused by the novel SARS-CoV-2 has so far claimed more than 60,000 lives and has impacted human lives worldwide. However, as the novel SARS-CoV-2 displays high transmission rates, their underlying genomic severity is required to be fully understood. We studied the complete genomes of 95 SARS-CoV-2 strains from different geographical regions worldwide to uncover the pattern of the spread of the virus. We show that there is no direct transmission pattern of the virus among neighboring countries suggesting that the outbreak is a result of travel of infected humans to different countries. We revealed unique single nucleotide polymorphisms (SNPs) in nsp13-16 (ORF1b polyprotein) and S-Protein within 10 viral isolates from the USA. These viral proteins are involved in RNA replication, indicating highly evolved viral strains circulating in the population of USA than other countries. Furthermore, we found an amino acid addition in nsp16 (mRNA cap-1 methyltransferase) of the USA isolate (MT188341) leading to shift in amino acid frame from position 2540 onwards. Through the construction of SARS-CoV-2-human interactome, we further revealed that multiple host proteins (PHB, PPP1CA, TGF-{beta}, SOCS3, STAT3, JAK1/2, SMAD3, BCL2, CAV1 & SPECC1) are manipulated by the viral proteins (nsp2, PL-PRO, N-protein, ORF7a, M-S-ORF3a complex, nsp7-nsp8-nsp9-RdRp complex) for mediating host immune evasion. Thus, the replicative machinery of SARS-CoV-2 is fast evolving to evade host challenges which need to be considered for developing effective treatment strategies.

microbiology↗

bioRxiv2020

Bacillus anthracis chain length, a virulence determinant, is regulated by a transmembrane Ser/Thr protein kinase PrkC

Anthrax is a zoonotic disease caused by Bacillus anthracis, a spore-forming pathogen that displays a chaining phenotype. It has been reported that in a mouse infection model, systemic inoculation with longer bacterial chains caused blockade in lung capillaries. The blockade resulted in increased pathophysiological consequences viz, hypoxia and lung tissue injury. Hence, chaining acts as a virulence factor and molecules that regulate the chaining phenotype can be the potential drug targets. In this study, we have identified the serine/threonine protein kinase of B. anthracis, PrkC, localized at the bacteria-host interface, as a determinant of bacterial chain length. In vitro, prkC disruption strain (BAS {Delta}prkC) grew as shorter chains throughout the bacterial growth cycle as observed through phase-contrast and scanning electron microscopy. Since molecules such as BslO, a septal murein hydrolase, that catalyzes daughter cell separation and Sap, an S-layer structural protein required for the septal localization of BslO, are known to influence chain length, a comparative analysis to determine their levels was done through western-blot analysis. Both BslO and Sap were found to be upregulated in BAS {Delta}prkC at the majority of the time points. Additionally, PrkC disruption was observed to have a significant effect on bacterial growth and cell wall thickness. In BAS {Delta}prkC strain, a decrease in the cell wall thickness and an increase in the multi-septa formation was observed through transmission electron and confocal microscopy respectively. Altogether, we show that PrkC disruption affected chaining phenotype, cell growth and cell wall thickness and also report that the associated molecules were de-regulated. Through this work, we show for the first time that the chaining phenotype is regulated by PrkC, a transmembrane kinase with a sensor domain. During infection, PrkC may regulate the chaining phenotype through the identified signaling mechanism. Authors summaryB. anthracis, a spore-forming pathogen is the causative agent of anthrax, a zoonotic disease that primarily affects livestock and wildlife. Humans are at risk of contracting this disease through exposure to spores generated by infected animals. In the past, B. anthracis spores have been used as a bioterror agent. Hence, there has been a continuous effort to understand the biology of this pathogen to develop both therapeutic and prophylactic treatment. Various virulence factors that are essential for B. anthracis pathogenesis have been identified. The ability of B. anthracis to grow in chains acts as a virulence factor. Longer bacterial chains are reported to cause blockade of lung capillaries in the mouse infection model. In this study, we have shown that the disruption of the lone serine/threonine protein kinase, PrkC, localized at the bacteria-host interface leads to the shortening of the bacterial chains. We have seen that the depletion of PrkC results in an increase in the levels of the proteins responsible for de-chaining. Also, we have analyzed the effect of the disruption on cell growth, bacterial cell wall and septa formation. Since PrkC is a surface localized kinase with an extracellular domain that lacks homology to human proteins, it can be a target for new drugs. Disruption of PrkC activity and hence the longer chains in vivo may prevent pathophysiological consequences associated with the capillary blockade.

microbiology↗

bioRxiv2019

DJ-1 (Park7) affects the gut microbiome, metabolites, and development of Innate Lymphoid Cells (ILCs)

The proper communication between gut and brain is pivotal for maintenance of health and dysregulation of the gut-brain axis can lead to several clinical disorders. Also, in Parkinsons disease (PD) 85% of all patients experienced constipation long before showing any signs of motor phenotypes. For differential diagnosis and when it comes to preventive treatment there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperon protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 modifies the PD risk remains incompletely understood. In the present study we provide evidence that DJ-1 is implicated in shaping the gut microbiome including their metabolite production or innate immune cells (ILCs) development. We revealed that in 4 months old mice genetic deficiency of DJ-1 leads to significantly decrease in several bacterial genera and significantly increase in two specific genera, namely Alistipes and Rikenella. DJ-1 deficient mice have a higher production of calprotectin/MCP-1 inflammatory protein - a known protein involved in colonic inflammation - and significantly higher expression of glial fibrillary acidic protein (GFAP) than control littermates. Expression of a-Synuclein, a key protein in Lewy bodies, in the colon was not significantly different between genotypes. Metabolic profiles of feces extracts analysed by H1-NMR spectroscopy showed increased short chain fatty acids (SCFAs) and decreased amino acid levels, suggesting a general switch from protein towards fibre degrading strains in DJ-1 deficient mice. We observed that Malonate - which is known to influence the immune system - has significantly higher concentrations in DJ-1 deficient mice. Moreover, DJ-1 deficient mice have high levels of the phenol derivate 3-(3-Hydroxyphenyl) propanoic acid (3-HPPA) which is a breakdown product of aromatic substrates like tyrosine, phenylalanine and polyphenols. DJ-1 deficient mice also showed significantly reduced percentage of ILCs. Thus, our data suggests that absence of DJ-1 leads to increase in gut inflammatory bacteria composition, deregulated metabolites and dysregulated innate immunity which could be a key factor in the initiation of PD disease in the gut, and potentially also in brain during disease progression.

immunology↗

bioRxiv2019

Enriched environmental conditions modify the gut microbiome composition and fecal markers of inflammation in Parkinson’s disease

BackgroundRecent findings suggest an implication of the gut microbiome in Parkinsons disease patients. Parkinsons disease onset and progression has also been linked with various environmental factors such as physical activity, exposure to pesticides, head injury, nicotine, and dietary factors.\n\nObjectivesIn this study, we used a transgenic mouse model overexpressing the complete human SNCA genes modeling familial and sporadic forms of Parkinsons disease to study whether environmental conditions such as standard versus enriched environment changes the gut microbiome and influences disease progression.\n\nMethodsWe performed 16S rRNA DNA sequencing on fecal samples for microbiome analysis and studied fecal inflammatory calprotectin from the colon of control and transgenic mice kept under standard environment and enriched environment conditions.\n\nResultsThe overall composition of the gut microbiota was not changed in transgenic mice compared with wild-type in enriched environment, however, individual gut bacteria at genus level such as Lactobacillus sp. were significantly changed in transgenic mice. Furthermore, enriched environment significantly reduced colon fecal inflammatory calprotectin protein in wild-type and transgenic enriched environment conditions compared to standard environment.\n\nConclusionOur data suggest that enriched social environment has a positive effect on the induction of SNCA mediated inflammation in the intestine by changing anti-inflammatory gut bacteria.

neuroscience↗

bioRxiv2018

Gut bacterial metabolite Urolithin A (UA) mitigates Ca2+ 1 entry inT cells by regulating miR-10a-5p

The gut microbiota influences several biological functions including immune response. Inflammatory bowel disease is favourably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolises ellagic acid leading to formation of bioactive urolithins A, B, C and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, how gut metabolite UA affects the function of immune cells remained incompletely understood. T cell proliferation is stimulated by store operated Ca2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4+ T cells with UA (10 {micro}M, 3 days) significantly blunted SOCE in CD4+ T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4+ T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4+ T cells. UA further decreased CD4+ T cell proliferation. Thus, bacterial metabolite UA up-regulates miR-10a-5p thus interfering with Orai1/STIM1/STIM2 expression, store operated Ca2+ entry and proliferation of murine CD4+ T cells.

immunology↗