10 research outputs found
Performance of a new Candida anti-mannan IgM and IgG assays in the diagnosis of candidemia
Candida is one of the most frequent pathogens of bloodstream infections, which is associated with high morbidity and mortality rates. Rapid immunological detection methods are essential in the early diagnosis of candidemia. Anti-mannan is one of host-derived biomarkers against cell wall components of Candida. We conducted this study to evaluate the diagnostic performance of two anti-mannan assays (IgM, IgG) for candidemia through the analysis of 40 candidemia patients, 48 participants with Candida colonization and 213 participants with neither Candida colonization nor Candida infections (13 patients with other bloodstream infections, 145 hospitalized patients and 55 healthy controls). The performance of the two assays were evaluated by calculating their sensitivity and specificity. The sensitivity ranged from 0.78 to 0.80 for the IgM assay and 0.68 to 0.75 for the IgG assay. The specificity ranged from 0.97 to 0.98 for the IgM assay and 0.91 to 0.94 for the IgG assay. The diagnostic performance of the anti-mannan IgM assay was better than that of IgG, with higher sensitivity and specificity. Combining the two assays (positive results of single or both assays are both considered as positive) could improve the sensitivity up to 0.93 (0.79-0.98) and only slightly reduce the specificity (0.93(0.89-0.95)). The anti-mannan IgM, IgG assays are rapid and cost-effective assays that may be probably useful in the diagnosis of candidemia
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout
Two of the main problems of stem cell and regenerative medicine are the exit of pluripotency and differentiation to functional cells or tissues. The answer to these two problems holds great value in the clinical translation of stem cell as well as regenerative medicine research. Although piling researches have revealed the truth about pluripotency maintenance, the mechanisms underlying pluripotent cell self-renewal, proliferation, and differentiation into specific cell lineages or tissues are yet to be defined. To this end, we took full advantage of a novel technology, namely, the genome-scale CRISPR-Cas9 knockout (GeCKO). As an effective way of introducing targeted loss-of-function mutations at specific sites in the genome, GeCKO is able to screen in an unbiased manner for key genes that promote exit from pluripotency in mouse embryonic stem cells (mESCs) for the first time. In this study, we successfully established a model based on GeCKO to screen the key genes in pluripotency withdrawal. Our strategies included lentiviral package and infection technology, lenti-Cas9 gene knockout technology, shRNA gene knockdown technology, next-generation sequencing, model-based analysis of genome-scale CRISPR-Cas9 knockout (MAGeCK analysis), GO analysis, and other methods. Our findings provide a novel approach for large-scale screening of genes involved in pluripotency exit and offer an entry point for cell fate regulation research
PPM1K mediates metabolic disorder of branched-chain amino acid and regulates cerebral ischemia-reperfusion injury by activating ferroptosis in neurons
Abstract Ischemic stroke is a neurological disorder caused by vascular stenosis or occlusion, accounting for approximately 87% of strokes. Clinically, the most effective therapy for ischemic stroke is vascular recanalization, which aims to rescue neurons undergoing ischemic insults. Although reperfusion therapy is the most effective treatment for ischemic stroke, it still has limited benefits for many patients, and ischemia-reperfusion (I/R) injury is a widely recognized cause of poor prognosis. Here, we aim to investigate the mechanism of protein phosphatase Mg2+/Mn2+ dependent 1 K (PPM1K) mediates metabolic disorder of branched-chain amino acids (BCAA) by promoting fatty acid oxidation led to ferroptosis after cerebral I/R injury. We established the I/R model in mice and used BT2, a highly specific BCAA dehydrogenase (BCKD) kinase inhibitor to promote BCAA metabolism. It was further verified by lentivirus knocking down PPM1K in neurons. We found that BCAA levels were elevated after I/R injury due to dysfunctional oxidative degradation caused by phosphorylated BCKD E1α subunit (BCKDHA). Additionally, the level of phosphorylated BCKDHA was determined by decreased PPM1K in neurons. We next demonstrated that BCAA could induce oxidative stress, lipid peroxidation, and ferroptosis in primary cultured cortical neurons in vitro. Our results further showed that BT2 could reduce neuronal ferroptosis by enhancing BCAA oxidation through inhibition of BCKDHA phosphorylation. We further found that defective BCAA catabolism could induce neuronal ferroptosis by PPM1K knockdown. Furthermore, BT2 was found to alleviate neurological behavior disorders after I/R injury in mice, and the effect was similar to ferroptosis inhibitor ferrostatin-1. Our findings reveal a novel role of BCAA in neuronal ferroptosis after cerebral ischemia and provide a new potential target for the treatment of ischemic stroke