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

Proton magnetic resonance metabolomic characterization of ovarian serous carcinoma effusions: chemotherapy-related effects and comparison with malignant mesothelioma and breast carcinoma

Malignant serous effusions are a common manifestation of advanced cancer, associated with significant morbidity and mortality. The aim of this study was to identify the metabolic differences between ovarian serous carcinoma effusions obtained pre- and post-chemotherapy, as well as to compare ovarian carcinoma (OC) effusions with breast carcinoma and malignant mesothelioma specimens. The supernatants of 115 effusion samples were analyzed by high-resolution magnetic resonance spectroscopy in vitro and multivariate analysis. The samples comprised pleural and peritoneal effusions from 95 OC, 10 breast carcinomas, and 10 malignant mesotheliomas. Among the OC, 8 were paired peritoneal specimens obtained pre- and post-chemotherapy from the same patient. OC had elevated levels of ketones (aceto-acetate and β-hydroxybutyrate) and lactate compared to malignant mesotheliomas and breast carcinomas, whereas the latter had more glucose, alanine, and pyruvate. Multivariate analysis of paired effusions in OC showed a significant increase in glucose and lipid levels in the post-treatment spectra (P = .039). Magnetic resonance spectroscopy is a promising technique for comprehensive and comparative studies of metabolites in malignant serous effusions, and our study shows that small metabolites associated with effusions might improve our understanding of tumor biology and disease progression and has diagnostic potential in this differential diagnosis

Activity and clinical relevance of autotaxin and lysophosphatidic acid pathways in high-grade serous carcinoma

The aim of this study was to analyze the expression, biological role and clinical relevance of autotaxin (ATX), the enzyme synthetizing lysophosphatidic acid (LPA), and LPA receptors (LPAR) in high-grade serous carcinoma (HGSC). mRNA expression by qRT-PCR of LPAR1-6 was analyzed in 155 HGSC specimens (88 effusions, 67 solid lesions). ATX mRNA expression was analyzed in 97 specimens. ATX, ERK, and AKT protein expression was studied by Western blotting. LPAR2 mRNA was overexpressed in HGSC cells in effusions compared to solid lesions, with opposite findings for LPAR3 and LPAR6 mRNA and ATX protein. Higher LPAR1 levels were significantly related to longer overall survival (OS) in pre-chemotherapy effusions (p = 0.027). Conversely, higher expression of LPAR1, LPAR2, and LPAR5 in post-chemotherapy effusions was significantly associated with shorter OS (p = 0.037, p = 0.025 and p = 0.021, respectively) and progression-free survival (PFS) (p < 0.001, p = 0.007 and p < 0.001, respectively) in univariate survival analysis. LPAR1 mRNA expression was an independent prognosticator of OS in patients with pre-chemotherapy effusions and PFS in patients with post-chemotherapy effusions (p = 0.013 both). In conclusion, LPAR mRNA and ATX protein levels are anatomic site-dependent in HGSC and the former are informative of disease outcome