In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing.

669 Application of In-cell Western Blot to Study Burn-induced Cardiac Dysfunction

Introduction Traditional Western blotting (WB) may not reflect true target protein amount due to interference from enzymic activity during sample preparation. In-Cell Western (ICW) blotting has proved to have advantages over the traditional WB. However, ICW has not been used to study burn-induced cardiac dysfunction. This study will explore the efficacy of ICW in the study of burn-induced cardiac dysfunction. Methods Human cardiomyocytes (Ac16) were cultured in serum from rats with/without 60% of total body surface area (TBSA) burn injury. Cultured cells were permeabilized by Tritons-100 X, 1st anti-targeted protein antibodies were added and incubated, and, finally, two fluorescence-conjugated 2nd antibodies (one red and one blue) were added and incubated. LI-COR Odyssey CLx scanner was employed to obtain images and LI-COR Image Studio 4.0 software was used to analyze image and GraphPad Prism 8.0 software was used to determine image density. Results ICW proved to be a good tool to study burn injury. Our ICW data showed that burn serum resulted in 1) increased cardiomyocyte PARP1 (leading to increased protein acetylation); 2) increased damage of cardiomyocyte mitochondrial DNA replication (via increase in mitochondrial Fis1and BCBN proteins and decrease in parkin and POLG proteins); and 3) a decrease of cardiomyocyte mitochondrial biogenesis (with increase in inflammation-related proteins, apoptosis-related proteins, and decrease Brf2-ARE-related proteins). Conclusions This study provides preliminary evidence that ICW may be a novel tool to study burn injury and confirms that burn-injury induced cardiomyocyte mitochondrial dysfunction occurs via damage of mitDNA replication, involving inflammation, apoptosis, and mitochondrial biogenesis pathways.

Lepidiumuridine A: A New Natural Uridine Derivative as a Phytoestrogen Isolated from the Seeds ofLepidium apetalumWilld.

There has been great interest in phytoestrogens, which are polyhydric compounds that are derived from plants and have a structure similar to that of the mammalian steroid hormone 17β-estradiol. The present study examined the estrogenic effects of a new natural uridine derivative, lepidiumuridine A (LA), that was isolated from the seeds ofLepidium apetalum. The structure was clarified and determined via analysis of extensive spectroscopic data interpretation. The activity of LA was investigated by measuring the levels of estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), and the uterus growth in mice. The proliferation experiment of MCF-7 breast cancer cells was also conducted. Western blot, in-cell western, and antagonist assays with methyl piperidino-pyrazole (MPP) were used for exploring the mechanism of the effects of LA. The results showed that LA elevated the uterine coefficient, the levels of E2, and FSH significantly. In addition, LA significantly elevated ERαexpression in the uterus and MCF-7 cells. MPP inhibited the proliferation of LA-stimulated MCF-7 cell and ERαexpression in MCF-7 cells. Taken together, LA had an estrogen-like effect, which was mainly mediated by the estrogen receptor ERα.

Kinetic effect of oestrogen on secretion of prostaglandins E2 and F2α in bovine oviduct epithelial cells

This study aimed to investigate the effect of oestrogen on prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) secretion in bovine oviduct epithelial cells. Bovine oviduct epithelial cells were obtained from the lumen of fresh bovine oviducts. Quantitative real-time polymerase chain reaction and in-cell western assays were used to measure PGE2 and PGF2α synthase activity and enzyme-linked immunosorbent assays were used to detect the concentrations of the two prostaglandins in extracellular fluid. We observed that oestradiol caused a short-term increase in cyclo-oxygenase-2 (COX-2), which stimulated PGE2 and PGF2α secretion, and that a subsequent decrease in COX-2 and an increase in cyclo-oxygenase-1 (COX-1) produced a high PGE2 : PGF2α ratio. These findings reflect the dynamic change in PGE2 and PGF2α levels under the influence of oestrogen, which may be essential for fertilisation.