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

A novel reliability assessment method for distribution networks based on linear programming considering distribution automation and distributed generation

Abstract The primary methods of assessing the reliability of distribution networks comprise analytic and simulation methods. However, both approaches require the identification and computation of network topology, which precludes their expression in explicit, continuous functions, consequently impeding the incorporation of reliability constraints into planning and operational optimization models. To tackle this restriction, the present work puts forth a novel linear‐programming‐based reliability assessment method that is mathematically formulated, considering distribution automation (DA) and distributed generations (DGs), consisting of both conventional and renewable energy sources. In this paper, the clustering method and the scenario‐based method are used to model DGs. Next, a mixed integer linear programming (MILP) model, considering the DA and DGs with the System Average Interruption Duration Index (SAIDI) as the optimization objective, is proposed. Finally, the feasibility and effectiveness of the proposed method are verified in a 37‐node distribution network system

Islet-1 may function as an assistant factor for histone acetylation and regulation of cardiac development-related transcription factor Mef2c expression.

OBJECTIVE:Islet-1 is an important transcription factor for cardiac development through mediating extensive interactions between DNA and proteins. The present study was to investigate the role of Islet-1 in regulating the expression of cardiac development-related transcription factors and mechanism. METHODS AND RESULTS:The expression of Islet-1 and histone acetylases (HATs) subtype p300 was determined in newborn mouse hearts and mouse embryonic hearts at different development stages using Western blot. The expression of Islet-1 and cardiac development-related transcription factors Mef2c, GATA4 and Tbx5 as well as histone H3 acetylation level were determined in cardiac progenitor cells with and without transfection of Islet-1 interference RNA (RNAi) in lentivirus using PCR and Western blot. Islet-1 peak expression occurred on day E14.5 in mouse embryonic heart, and was present in the promoter regions of Mef2c, GATA4 and Tbx5 that were precipitated with p300 antibody. When Islet-1 was inhibited with specific RNAi in cardiac progenitor cells, the expression of Mef2c and Tbx5, but not GATA4, was significantly suppressed along with selective reduction in histone H3 acetylation in the promoter region of Mef2c, but not GATA4 and Tbx5. The level of Mef2c DNA, not GATA4 and Tbx5, in the complex associated with p300 was significantly decreased in the cells with Islet-1 knockdown. CONCLUSIONS:These data suggested that Islet-1 might function as an assistant factor that was involved in the regulation of histone acetylation and Mef2c expression via assisting p300 on specifically targeting the promoter of Mef2c

Increased CD8+CD28+ T cells independently predict better early response to stereotactic ablative radiotherapy in patients with lung metastases from non-small cell lung cancer

Abstract Background Stereotactic ablative radiotherapy (SABR) shows a remarkable local control of non-small cell lung cancer (NSCLC) metastases, partially as a result of host immune status. However, the predictors of immune cells for tumor response after SABR are unknown. To that effect, we investigated the ability of pre-SABR immune cells in peripheral blood to predict early tumor response to SABR in patients with lung metastases from NSCLC. Methods This study included 70 patients with lung metastases from NSCLC who were undergoing SABR. We evaluated the early tumor response 1 month and 6 months after SABR in these patients following RECIST 1.1 guidelines. Pre-SABR peripheral CD8+ T cell count, CD8+CD28+ T-cell count, CD8+CD28− T-cell count, CD4+ T-cell count, and Treg-cell count were measured using flow cytometry. Results Increased CD8+CD28+ T-cell counts (14.43 ± 0.65 vs. 10.21 ± 0.66; P = 0.001) and CD4/Treg ratio (16.96 ± 1.76 vs. 11.91 ± 0.74; P = 0.011) were noted in 1-month responsive patients, compared with non-responsive patients. In univariate logistic analyses, high CD8+CD28+ T-cell counts (OR 0.12, 95% CI 0.03–0.48; P = 0.003), CD4/Treg ratio (OR 0.24, 95% CI 0.06–0.90; P = 0.035), and BED10 (OR 0.91, 95% CI 0.84–0.99; P = 0.032) predicted a 1-month tumor response to SABR. According to multivariate logistic analyses, the CD8+CD28+ T-cell count predicted a 1-month tumor response to SABR (OR 0.19, 95% CI 0.04–0.90; P = 0.037) independently. Furthermore, we confirmed the independent predictive value of the CD8+CD28+ T-cell count in predicting tumor response to SABR in 41 patients 6 months after treatment (OR 0.08, 95% CI 0.01–0.85; P = 0.039). Conclusions A pre-SABR CD8+CD28+ T-cell count could predict early tumor response to SABR in patients with lung metastases from NSCLC. Larger, independently prospective analyses are warranted to verify our findings

Expression of Islet-1 protein in mouse embryonic and neonatal hearts.

<p>A representative Western blotting showed that Islet-1 was dynamically expressed in the embryonic hearts. The expression of Islet-1 in mouse embryonic hearts was significantly higher on E14.5 than that on E11.5, E17.5, and neonatal mice. * <i>p</i> < 0.05 (n=3).</p

Acetylation level of histone H3 in cardiac progenitor cells after inhibiting Islet-1 expression.

<p>Western blotting analysis showed that detectable level of histone H3 acetylation was present in cardiac progenitor cells, and was not significantly changed when Islet-1 expression was inhibited (A, <i>p</i>>0.05, n=3). However, the DNA quantity of Mef2c in the promoter region immunoprecipitated with acH3 antibody was significantly decreased in the cells transfected with Islet-1 RNAi as compared with the controls, while no difference was observed in the DNA quantity of GATA4 and Tbx5 in the cells (B). *<i>p</i><0.05, n=3. </p

Transcriptional expression of cardiac development-related transcription factors in cardiac progenitor cells after lentivirus transfeciton with Islet-1 RNAi.

<p>Quantitative RT-PCR analysis showed the mRNA level for Islet-1 was dramatically reduced in cardiac progenitor cells when transfected with Islet-1 RNA interference vector to inhibit Islet-1 (A). The mRNA levels for Mef2c and Tbx5 were significantly reduced in the cells when Islet-1 expression was inhibited with RNAi as compared with the controls. There was no significant difference in GATA4 mRNA level in the cells with Islet-1 knockdown with RNAi (B). *<i>p</i> < 0.05, n=3-5. </p

Interaction of p300 with the promoter regions of Mef2c, GATA4 and Tbx5 in cardiac progenitor cells.

<p>The DNA quantity of Mef2c in the promoter regions immunoprecipitated using p300 antibody was significantly decreased in the cells transfected with Islet-1 RNAi as compared with the controls. However, no difference in the DNA quantity of GATA4 and Tbx5 was observed in the cells with or without transfection with Islet-1 RNAi. * <i>p</i> < 0.05, n=3.</p