Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Culturing of primary bovine mammary epithelial cells and validation of biotransformation capacity in experiments with enrofloxacin

Many drugs and toxic compounds are subjected to disposition and metabolism in bovine mammary epithelial cells (bMECs). For rapid investigation of different compounds and their possible interactions, validated in vitro models are needed. Therefore, the first objective of described experiments was to develop the techniques for cell isolation, purification and culturing of bMECs. The second objective was the application of these cell cultures in a well-known substrate for one of the major biotransformation enzymes in epithelial cells. To this end, the metabolism of enrofloxacin (ENR) into its active metabolite ciprofloxacin (CPR), was studied. This conversion is known to be catalysed by enzymes of the cytochrome P4501A and P4503A family. The expression profile of these enzymes shows a close correlation with cellular ABC-efflux transporters. Primary bMECs were isolated from healthy udders of lactating cows (n=5 animals). mRNA levels of α-casein, b-lactoferrin and cyclophilin B were determined as markers of cell identity of purity of the cultures. Subsequently, bMECs cultures were incubated with ENR (10 µM). Concentrations of ENR and its main metabolite CPR in the medium and in the cells were determined by HPLC-FL analysis. Gene expression of CYP1A1, CYP1A2 and CYP3A4, bovine ABCG2 was detected by qRT-PCR. Results showed that ENR penetrated into bMECs and was converted to CPR. CPR was excreted in the medium suggesting participation of ABCG2 in fluoroquinolone efflux. In conclusion, the data showed that the established bMEC cultures expressed major CYP450 enzymes as well as the most relevant efflux transport ABGG2. This model should be further validated and can serve as an interesting model for further studies on site-specific drug/toxin metabolism and transport in the bovine mammary gland.

Identification of Salmonella Typhimurium Peptidyl-prolyl cis-trans Isomerase B (PPIase B) and Assessment of their Role in the Protein Folding

Background: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. Objective: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. Methods: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. Result: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/μg, Km = 0.6219 ± 0.1701 μM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. Conclusion: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.

CypB-CD147 Signaling Is Involved in Crosstalk between Cartilage and FLS in Collagen-Induced Arthritis

To investigate the crosstalk between cartilage and fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), we adopted an in vitro coculture system model of collagen-induced arthritis (CIA) cartilage and CIA FLS monolayer. CIA rat samples of the synovium and femur head were collected for isolation of FLS and coculture system. Cartilages were treated with vehicle (Ctrl group), 10 ng/mL interleukin- (IL-) 1α (IL-1α group), and 10 ng/mL IL-1α plus 10 μM dexamethasone (Dex group) for 3 days before coculture with FLS for further 2 days. After the coculture, FLS were collected to determine the influences of articular cartilage on synoviocytes. Whether the CypB-CD147 signaling pathway is involved in the interactions between cartilage and FLS is assayed. Results showed that IL-1α-stimulated CIA cartilage promoted the proliferation and reduced the apoptosis of FLS. Increased inflammatory cytokines and decreased p57 expression were found in cocultured FLS stimulated by IL-1α-challenged CIA cartilage. Upregulation of NF-κB and I-κB kinase β (IKK-β) and downregulation of the inhibitor of NF-κBα (I-κBα) protein were observed in cocultured FLS. After coculture, significant increases in the expression of cyclophilin B (CypB) and CD147 were observed in CIA cartilage and FLS, respectively. Furthermore, results of immunofluorescence staining showed that the anti-CD147 antibody significantly suppressed p65 nuclear translocation in cocultured FLS stimulated by IL-1α-challenged CIA cartilage. In conclusion, inflammatory effects in the cartilage-FLS coculture system are associated with the CypB-CD147 mediating NF-κB pathway which may further enhance the inflammation in RA.