Biology subjects

Ghimire, S.

Publications and source records attributed to Ghimire, S..

48 records · Page 3Linked to original sources

bioRxiv2019

When two cells are better than one: specialized stellate cells provide a privileged route for uniquely rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 members of the Major Intrinsic Protein family are expressed at very high level in the fly renal tissue; the aquaporins Drip and Prip, and the aquaglyceroporins Eglp2 and Eglp4. As predicted from their structure and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes impacts tubule performance resulting in impaired hormone-induced fluid secretion. The Drosophila tubule has two main secretory cell types: active cation-transporting principal cells with the aquaglyceroporins localize to opposite plasma membranes and small stellate cells, the site of the chloride shunt conductance, with these aquaporins localising to opposite plasma membranes. This suggests a model in which cations are pumped by the principal cells, causing chloride to follow through the stellate cells in order to balance the charge. As a consequence, osmotically obliged water follows through the stellate cells. Consistent with this model, fluorescently labelled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects.\n\nSignificance statementThe tiny insect renal (Malpighian) tubule can transport fluid at unparalleled speed, suggesting unique specialisations. Here we show that strategic allocation of Major Intrinsic Proteins (MIPs) to specific cells within the polarized tubule allow the separation of metabolically intense active cation transport from chloride and water conductance. This body plan is general to at least many higher insects, providing a clue to the unique success of the class Insecta.

physiology↗

bioRxiv2019

The Gut Microbiota composition of Feral and Tamworth Pigs determined using High-Throughput Culturomics and Metagenomics Reveals Compositional Variations When Compared to the Commercial Breeds

Bacterial communities in the hindguts of pigs have a profound impact on health and disease. Yet very limited studies have been performed outside intensive swine farms to determine pig gut microbiome composition in natural populations. Feral pigs represent a unique situation where the microbiome structure can be observed outside the realm of modern agriculture. Additionally, Tamworth pigs that freely forage were included to characterize the microbiome structure of this rare breed. In this study, gut microbiome of feral and Tamworth pigs were determined using metagenomics and culturomics. Tamworth pigs are highly dominated by Bacteroidetes primarily composed of the genus Prevotella whereas feral samples were more diverse with almost equal proportions of Firmicutes and Bacteroidetes. In total, 46 distinct species were successfully isolated from 1000 colonies selected. The combination of metagenomics and culture techniques facilitated a greater retrieval of annotated genes than either method alone. Furthermore, the naturally raised Tamworth pig microbiome contained more number of antibiotic resistance genes when compared to feral pig microbiome. The single medium based pig microbiota library we report is a resource to better understand pig gut microbial ecology and function by assembling simple to complex microbiota communities in bioreactors or germfree animal models.

microbiology↗

bioRxiv2019

Description of a new member of the family Erysipelotrichaceae: Clostridium fusiformis sp. nov., isolated from healthy human feces

A Gram-positive, non-motile, rod-shaped facultative anaerobic bacterial strain SG502T was isolated from the healthy human fecal samples in Brookings, SD, USA. The comparison of the 16S rRNA gene placed the strain within the Clostridium cluster XVI, where, Clostridium innocuum ATCC 14501T, Longicatena caecimuris strain PG-426-CC-2, Eubacterium dolichum DSM 3991T and Eubacterium tortuosum DSM 3987T were its closest taxa with 95.15%, 94.49%, 93.28%, and 93.20% sequence identities respectively. The optimal growth temperature and pH for the strain SG502T were 37{degrees}C and 7.0 respectively. Acetate was the major short-chain fatty acid product of the strain SG502T when grown in BHI-M medium. The major cellular fatty acids produced by the strain SG502T were C18:1 {omega}9c, C18:0 and C16:0. The DNA G+C content of the strain was 34.34 mol%. The average nucleotide identity of the genome of the strain SG502T and its closest neighbor C. innocuum ATCC 14501T was 63.48%. Based on the polyphasic analysis, the type strain SG502T (=DSM 107282T), represents a novel species of the genus Clostridium for which the name Clostridium fusiformis sp. nov. is proposed.

microbiology↗

bioRxiv2019

Sellimonas caecigallum sp. nov., description and genome sequence of a new member of the Sellimonas genus isolated from the cecum of feral chicken

An obligately anaerobic, non-motile, Gram-positive coccobacillus strain SW451 was isolated from pooled cecum contents of feral chickens. Comparative analysis based on 16s rRNA sequence showed that strain SW451 had 95.24% nucleotide sequence similarity to Sellimonas intestinalis BR31T, the closest species with a valid taxonomy. The genome of SW451 is 2.67 Mbp with 45.23 mol% of G+C content. The major cellular fatty acids were C16: 0, C14: 0 and C16: 0 DMA. Based on taxonogenomic, physiological, and biochemical analysis, the strain SW451 represents a new species of the genus Sellimonas, for which the name Sellimonas caecigallum sp. nov. is proposed. The type strain of Sellimonas caecigallum is SW451 (=DSM 109473T)

microbiology↗

bioRxiv2019

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production

ObjectiveEnteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function, however, the role of Rfx6 in EECs differentiation remained to be elucidated.\n\nMethodsWe examined the molecular, cellular and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes.\n\nResultsRfx6 is expressed in the gut endoderm; later it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, whose differentiation programs are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1- or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine.\n\nConclusionThese studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

developmental biology↗

bioRxiv2019

Olsenella lakotia SW165 sp. nov., an acetate producing obligate anaerobe with a GC rich genome

A Gram-positive and obligately anaerobic bacterium was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. The microorganism grew at 37-45{degrees} C and pH 6-7.5. This strain produced acetic acid as the primary metabolic end product. Major fatty acids were C12:0, C14:0, C14:0 DMA and summed feature 1 (C13:1 at 12-13 and C14:0 aldehyde). Phylogenetic analyses based on 16S rRNA gene sequence suggested that strain SW165 belongs to the family Atopobiaceae with the closest relatives being Olsenella profusa DSM 13989T (96.33% similarity), Olsenella umbonate DSM 26220T (96.18%) and Olsenella uli DSM 7084T (96.03%). Genome sequencing revealed a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the highest G+C content among members of the genus Olsenella. Phylogenetic and phenotypic comparison indicated that strain SW165 represents a novel species of the genus Olsenella, for which the name Olsenella lakotia sp. nov. is proposed. The type strain is SW165 (=DSM 107283T).

microbiology↗

bioRxiv2018

Hepatic γ-Amino Butyric Acid Release Drives Hyperinsulinemia and Insulin Resistance

The degree of hepatic lipid accumulation in obesity correlates with the severity of hyperinsulinemia and systemic insulin resistance. Here, we propose a mechanism that explains this associative link, whereby, hepatic steatosis dysregulates glucose and insulin homeostasis. Obesity-induced hepatocellular lipid accumulation results in hepatocyte depolarization. We have established that hepatocyte depolarization depresses hepatic afferent vagal nerve firing, increases GABA release from liver slices, and causes hyperinsulinemia. Preventing hepatic GABA release or eliminating the ability of the liver to communicate to the hepatic vagal nerve ameliorates the hyperinsulinemia and insulin resistance associated with diet-induced obesity. In people with obesity hepatic expression of GABA transporters is associated with glucose infusion and disposal rates during a hyperinsulinemic euglycemic clamp. Single nucleotide polymorphisms in hepatic GABA re-uptake transporters are associated with an increased incidence of type II diabetes mellitus. Herein, we identify GABA as a novel hepatokine that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity.

physiology↗

bioRxiv2018

Gut Microbial succession during conventionalization of germfree chicken

A gnotobiotic chicken model was developed to study the succession of intestinal microflora from hatching to 18 days of age. Intestinal samples were collected from a local population of feral chickens and administered orally to germ-free 3 day old chicks. Animals were enthanized on 0, 9 and 18 days of age and intestinal samples were collected and subjected to genomic analysis. The five most prevalent phyla were Bacteroidetes (45.73{+/-}3.35%), Firmicutes (36.47{+/-}2.60%), Proteobacteria (8.28{+/-}0.91%), Actinobacteria (5.09{+/-}0.52%), and Spriochetes (2.10{+/-}0.38%). Principle coordinate analysis indicated the 0, 9 day and 18 day variables clustered together and the microbial communities changed temporally. The Morista-Horn index values ranged from 0.72 to 1, indicating the communities at 0, 9 or 18 days were more similar than dissimilar. The predicted functional profiles of the microbiomes of 0, 9 and 18 days were also similar. These results indicate the gnotobiotic chicks stably maintain the phylogentic diversity and predicted metabolic functionality of the inoculum community.\n\nImportanceThe domestic chicken is the cornerstone of animal agriculture worldwide with a flock population exceeding 40 billion birds/year. It serves as the economically valuable source of protein globally. Microbiome of poultry has important effects on chicken growth, feed conversion, immune status and pathogen resistance. The significance of our research is in developing a gnotobiotic chicken model to study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse set of gut bacteria. Therefore, besides using this model to study mechanisms of gut microbiota interactions in the chicken gut, our model could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.

microbiology↗

bioRxiv2018

Transmembrane Protein 135 (TMEM135) is a Liver X Receptor Target Gene that Mediates an Auxiliary Peroxisome Matrix Protein Import Pathway

The liver x receptors (LXRs) are key regulators of systemic lipid metabolism. We determined whether transmembrane protein 135 (TMEM135) is an LXR target gene and its physiologic function. An LXR agonist increased TMEM135 mRNA and protein in human hepatocyte and macrophage cell lines, which was prevented by LXR knockdown. The human TMEM135 promoter contains an LXR response element that bound the LXRs via EMSA and ChIP, and mediated LXR-induced transcription in reporter assays. Knockdown of TMEM135 in HepG2 cells caused triglyceride accumulation despite reduced lipogenic gene expression, indicating a potential role in {beta}-oxidation. To determine physiologic importance, TMEM135 was knocked-down via siRNA in livers of fed and fasted C57BL/6 mice. Fasting increased hepatic fatty acid and NADH concentrations in control mice, consistent with increased fatty acid uptake and {beta}-oxidation. However, in fasted TMEM135 knockdown mice, there was a further significant increase in hepatic fatty acid concentrations and a significant decrease in NADH, indicating an impairment in {beta}-oxidation by peroxisomes and/or mitochondria. Conversely, hepatic ketones tended to increase in fasted TMEM135 knockdown compared to control mice, and because ketogenesis is exclusively dependent on mitochondrial {beta}-oxidation, this indicates peroxisomal {beta}-oxidation was impaired in knockdown mice. Localization studies demonstrated that TMEM135 co-localized with peroxisomes but not mitochondria. Mechanistically, proteomic and Western blot analyses indicated that TMEM135 regulates concentrations of matrix enzymes within peroxisomes. In conclusion, TMEM135 is a novel LXR target gene in humans that mediates peroxisomal metabolism, and thus TMEM135 may be a therapeutic target for metabolic disorders associated with peroxisome dysfunction.

cell biology↗

bioRxiv2018

Genome sequence and description of Blautia brookingsii str SG772 nov., a new species of anaerobic bacterium isolated from healthy human gut

Strain SG-772 is a Gram positive, strictly anoxic bacterium isolated from the feces of a healthy human fecal donor. Based on 16S rRNA gene sequence, the strain showed maximum similarity (94.39%) with Blautia stercoris GAM6-1 in EZ-Taxon server and thus assigned the genus Blautia. The scanning electron micrograph of the bacterium revealed the characteristic coccobacillus shape as well as the complete absence of flagellum, suggesting its non-motile phenotype. This strain was found to utilize 27 substrates based on Biolog AN plates assay, with maximum preference for D-mannitol. Additionally, the strain was found to be resistant to tetracycline and streptomycin. Genome sequencing and analysis revealed an overall genome size of 3.49 Mbp and GC content of 43.97%. Based on RAST annotation server, the closest neighbor was Blautia hansenii DSM20583. Average Nucleotide Identity (ANI) of these strains were 81.69%, suggesting a high level of genomic variation. The comparative genome analysis of strain SG772 with B. hansenii DSM20583 revealed a total of 411 orthologous genes coding for basic metabolic functions. Furthermore, the genomes were functionally distinct based on COG categories. Thus, based on all these differences, we propose a novel species of genus Blautia named as Blautia brookingsii SG772.

microbiology↗

bioRxiv2017

Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife

The aim of this study was to generate a reference set of Salmonella enterica genomes isolated from wildlife from the United States and to determine the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. We sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pet cases that were submitted to the Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. 50.48% isolates showed resistance to at least one antibiotic. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we report here indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we report here could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis.

microbiology↗

bioRxiv2017

Metagenomic characterization of the effect of feed additives on the gut microbiome and antibiotic resistome of feedlot cattle

In North America, antibiotic feed additives such as monensin and tylosin are added to the finishing diets of feedlot cattle to counter the ill-effects of feeding diets with rapidly digestible carbohydrates. While these feed additives have been proven to improve feed efficiency, and reduce liver abscess incidence, how these products impact the gastrointestinal microbiota is not completely understood. Furthermore, there are concerns that antibiotic feed additives may expand the antibiotic resistome of feedlot cattle by enriching antimicrobial resistance genes in pathogenic and nonpathogenic bacteria in the gut microbiota. In this study, we analyzed the impact of providing antibiotic feed additives to feedlot cattle using metagenome sequencing of treated and untreated animals. Our results indicate that use of antibiotic feed additives does not produce discernable changes at the phylum level however treated cattle had reduced the abundance of gram-positive bacteria at the genus level. The abundance of Ruminococcus, Erysipelotrichaceae and Lachanospira in the gut of treated steers was reduced. This may impact the ability of these animals to exclude pathogens from the gut. However, our results did not show any correlation between the presence of antimicrobial resistance genes in the gut microbiota and the administration of antibiotic feed additives.

microbiology↗