Advances in microbial biotechnology of bile acids

The recent advances in microbial biotechnology of production of bile acid metabolites helped to identify a number of neutral and acidic steroidal compounds useful as drugs and drug intermediates on a scale which would not have been possible by classical chemical transformations. Microbial transformations viz., hydroxylation, dehydroxylation, reduction of the carbonyl moieties, epimerization, side-chain metabolism, introduction of carbon-carbon double bonds into the steroid nucleus, deconjugation of bile acid conjugates carried out by various microorganisms for production of useful metabolites with special reference to newer techniques including cell immobilization and transposon mutagenesis for selective transformations are reviewed. The different pathways of microbial degradation of bile acids leading to the formation of various products are discussed. A compilation of the metabolites formed by various microorganisms from the bile acids or their conjugates and reported during the period 1979-1992 is also provided.     

Determining clinically relevant features in cytometry data using persistent homology

Cytometry experiments yield high-dimensional point cloud data that is difficult to interpret manually. Boolean gating techniques coupled with comparisons of relative abundances of cellular subsets is the current standard for cytometry data analysis. However, this approach is unable to capture more subtle topological features hidden in data, especially if those features are further masked by data transforms or significant batch effects or donor-to-donor variations in clinical data. We present that persistent homology, a mathematical structure that summarizes the topological features, can distinguish different sources of data, such as from groups of healthy donors or patients, effectively. Analysis of publicly available cytometry data describing non-naïve CD8+ T cells in COVID-19 patients and healthy controls shows that systematic structural differences exist between single cell protein expressions in COVID-19 patients and healthy controls. We identify proteins of interest by a decision-tree based classifier, sample points randomly and compute persistence diagrams from these sampled points. The resulting persistence diagrams identify regions in cytometry datasets of varying density and identify protruded structures such as ‘elbows’. We compute Wasserstein distances between these persistence diagrams for random pairs of healthy controls and COVID-19 patients and find that systematic structural differences exist between COVID-19 patients and healthy controls in the expression data for T-bet, Eomes, and Ki-67. Further analysis shows that expression of T-bet and Eomes are significantly downregulated in COVID-19 patient non-naïve CD8+ T cells compared to healthy controls. This counter-intuitive finding may indicate that canonical effector CD8+ T cells are less prevalent in COVID-19 patients than healthy controls. This method is applicable to any cytometry dataset for discovering novel insights through topological data analysis which may be difficult to ascertain otherwise with a standard gating strategy or existing bioinformatic tools.     

Acute and subchronic toxicity of aflatoxin B1 to rohu, Labeo rohita (Hamilton)

Pathological alterations in various organs of rohu (L. rohita) fingerlings following acute (0, 7.50, 11.25 and 13.75 mg/kg body weight) and subchronic (0, 1.25 and 2.50 mg/kg body weight) single i.p. aflatoxin B1 exposure for 10 and 90 days, respectively, were investigated. Mortality (dose-dependent) was marked only during acute toxicosis. The changes observed in various organs were dose and time dependent. The acute dose groups revealed toxic changes viz., necrotic and vascular changes in liver and gill lamellae; meningitis, congestion in brain, degeneration and inflammatory reaction in heart along with degenerative to necrotic changes in kidney tubules and sloughing of the intestinal mucosa. During subchronic exposure to this toxin, preneoplastic lesions in liver along with changes in spleen, intestine, gill and pancreas were recorded. With low doses of aflatoxin, the fish did not reveal any mortality or external signs other than catchexia and increased pigmentation on scales. In composite culture practice of Indian major carps, this could be of economic significance.     

Clastogenic effects of dietary supplement--Spirulina alga, and some medicinal plant products from Boswellia serrata, Withania somnifera on mice

Pretreatment of aqueous extracts of Zyrulina (Spirulina), Aswagandha (Withania) and Nopane (Boswellia) on colchicine induced chromosome damage showed weakness of clastogenic activity in Swiss albino mice. None of the treatments increased significantly the number of chromosome aberrations.     

Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell

Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days. Bacteria were more susceptible than actinomycetes. Carbofuran and ethion were moderately toxic to soil microflora. Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran. GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days). GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp. (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp. (36.9 and 55.4%) and B. sphaericus (67.6% GDH) respectively. Carbofuran inhibited the enzyme activity in B. subtilis (55.55 and 35.3%) and to some extent in B. sphaericus. Ethion moderately inhibited LDH activity in Rhodococcus sp. AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E. coli HB 101 (34.2% LDH) as evidenced by formazan formation. From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility. It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes.     

Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.     

SopE acts as an Rab5-specific nucleotide exchange factor and recruits non-prenylated Rab5 on Salmonella-containing phagosomes to promote fusion with early endosomes

Rab-GTPase regulates the fusion between two specific vesicles. It is well documented that, for their biological function, Rab proteins need to be prenylated for attachment to the vesicle membrane. In contrast, we showed in the present investigation that SopE, a type III secretory protein of Salmonella, translocates onto Salmonella-containing phagosomes (LSP) and mediates the recruitment of non-prenylated Rab5 (Rab5:DeltaC4) on LSP in GTP form. Simultaneously, SopE present in infected cell cytosol acts as an Rab5-specific exchange factor and converts the inactive Rab-GDP to the GTP form. The non-prenylated Rab5 subsequently promoted efficient fusion of LSP with early endosomes. This is the first demonstration that a prenylation-deficient Rab protein retains biological activity and can promote vesicle fusion, if it is recruited on the membrane by some other method.     

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.