Effects of CYP inhibitors on precocene I metabolism and toxicity in rat liver slices

We present a comprehensive in vitro approach to assessing metabolism-mediated hepatotoxicity using male Sprague–Dawley rat liver slices incubated with the well characterized hepatotoxicant, precocene I, and inhibitors of cytochrome P450 (CYP) enzymes. This approach combines liquid chromatography mass spectrometry (LC MS) detection methods with multiple toxicity endpoints to enable identification of critical metabolic pathways for hepatotoxicity. The incubations were performed in the absence and presence of the non-specific CYP inhibitor, 1-aminobenzotriazole (ABT) and isoform-specific inhibitors. The metabolite profile of precocene I in rat liver slices shares some features of the in vivo profile, but also had a major difference in that epoxide dihydrodiol hydrolysis products were not observed to a measurable extent. As examples of our liver slice metabolite identification procedure, a minor glutathione adduct and previously unreported 7-O-desmethyl and glucuronidated metabolites of precocene I are reported. Precocene I induced hepatocellular necrosis in a dose- and time-dependent manner. ABT decreased the toxicity of precocene I, increased exposure to parent compound, and decreased metabolite levels in a dose-dependent manner. Of the isoform-specific CYP inhibitors tested for an effect on the precocene I metabolite profile, only tranylcypromine was noticeably effective, indicating a role of CYPs 2A6, 2C9, 2Cl9, and 2E1. With respect to toxicity, the order of CYP inhibitor effectiveness was ABT > diethyldithiocarbamate∼tranylcypromine > ketoconazole. Furafylline and sulfaphenazole had no effect, while quinidine appeared to augment precocene I toxicity. These results suggest that rat liver slices do not reproduce the reported in vivo biotransformation of precocene I and therefore may not be an appropriate model for precocene I metabolism. However, these results provide an example of how small molecule manipulation of CYP activity in an in vitro model can be used to confirm metabolism-mediated toxicity.     

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening

Salmonella enterica serovar Typhi and Typhimurium are closely related serovars. However, S. Typhi, a human-specific pathogen, has 5% of genes as pseudogenes, far more than S. Typhimurium, which only has 1%. One of these pseudogenes corresponds to sopD2, which in S. Typhimurium encodes an effector protein involved in Salmonella-containing vacuole biogenesis in human epithelial cell lines, which is needed for full virulence of the pathogen. We investigated whether S. Typhi trans-complemented with the functional sopD2 gene from S. Typhimurium (sopD2(STM) ) would reduce the invasion of human epithelial cell lines. Our results showed that the presence of sopD2(STM) in S. Typhi significantly modified the bacterial ability to alter cellular permeability and decrease the CFUs recovered after cell invasion of human epithelial cell line. These results add to mounting evidence that pseudogenes contribute to S. Typhi adaptation to humans.     

Diagnosis of Helicobacter pylori bacterial infections using a voltammetric biosensor

The voltammetric assay of Helicobacter pylori DNA was investigated using a bismuth-immobilized carbon nanotube electrode (BCNE). The analytical cyclic voltammetry (CV) peak potential was obtained at a 0.4 V reduction scan, where the diagnostic optimum square-wave (SW) stripping working range was achieved at 0.72-7.92 μg/mL H. pylori DNA (11 points). A relative standard deviation of 1.68% (RSD, n = 5) was obtained with 3.2 mg/mL H. pylori DNA using a 240 s accumulation time. Under optimum conditions, detection limit was 0.06 μg/mL. The developed sensors can be used for clinical application in the 15th doubted human gastric tissues, since the patient's peak current increased a hundred times more than the negative healthy tissue did. The sensing time obtained was only two minutes, and the process was simpler compared to common PCR amplification and electrophoresis photometric detection systems.     

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin‐producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin‐producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx‐related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx‐related events and enhance Stx absorption during STEC infection.     

Hyponatremia as a Predictor of Mortality in Peritoneal Dialysis Patients

Background and Aim

Hyponatremia is common in patients with chronic kidney disease and is associated with increased mortality in hemodialysis patients. However, few studies have addressed this issue in peritoneal dialysis (PD) patients.

Methods

This prospective observational study included a total of 441 incident patients who started PD between January 2000 and December 2005. Using time-averaged serum sodium (TA-Na) levels, we aimed to investigate whether hyponatremia can predict mortality in these patients.

Results

Among the baseline parameters, serum sodium level was positively associated with serum albumin (β = 0.145; p = 0.003) and residual renal function (RRF) (β = 0.130; p = 0.018) and inversely associated with PD ultrafiltration (β = −0.114; p = 0.024) in a multivariable linear regression analysis. During a median follow-up of 34.8 months, 149 deaths were recorded. All-cause death occurred in 81 (55.9%) patients in the lowest tertile compared to 37 (25.0%) and 31 (20.9%) patients in the middle and highest tertiles, respectively. After adjusting for multiple potentially confounding covariates, increased TA-Na level was associated with a significantly decreased risk of all-cause (HR per 1 mEq/L increase, 0.79; 95% CI, 0.73–0.86; p<0.001) and infection-related (HR per 1 mEq/L increase, 0.77; 95% CI, 0.70–0.85; p<0.001) deaths.

Conclusions

This study showed that hyponatremia is an independent predictor of mortality in PD patients. Nevertheless, whether correcting hyponatremia improves patient survival is unknown. Future interventional studies should address this question more appropriately.     

Annexin A2 Reduces PCSK9 Protein Levels via a Translational Mechanism and Interacts with the M1 and M2 Domains of PCSK9

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9''s C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.     

Psychedelic-inspired drug discovery using an engineered biosensor

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