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

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Expression of Partitioning Defective 3 (Par-3) for Predicting Extrahepatic Metastasis and Survival with Hepatocellular Carcinoma

Partitioning defective 3 (Par-3), a crucial component of partitioning-defective complex proteins, controls cell polarity and contributes to cell migration and cancer cell epithelial-to-mesenchymal transition. However, the clinical relevance of Par-3 in tumor progression and metastasis has not been well elucidated. In this study, we investigated the impact and association of Par-3 expression and clinical outcomes with hepatocellular carcinoma (HCC). We first confirmed that Par-3 was abundantly expressed in HCC cell lines by Western blot analysis. We used immunohistochemistry to analyze the association of Par-3 expression and clinicopathological characteristics in primary and subsequent metastatic tumors of patients with HCC. Par-3 was overexpressed in 47 of 111 (42.3%) primary tumors. Increased expression of Par-3 in primary tumors predicted an increased five-year cumulative incidence of extrahepatic metastasis. In addition, multivariate analysis revealed that Par-3 overexpression was an independent risk factor of extrahepatic metastasis. Increased Par-3 expression in primary tumors was associated with poor five-year overall survival rates and was an independent prognostic factor on Cox regression analysis. In conclusion, we show for the first time that increased Par-3 expression is associated with distant metastasis and poor survival rates in patients with HCC. Par-3 may be a novel prognostic biomarker and therapeutic target for HCC

14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

<div><p>Background</p><p>14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.</p> <p>Methodology and Principal Findings</p><p>In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.</p> <p>Significance</p><p>Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.</p> </div

14-3-3ε suppresses E-cadherin expression <i>via</i> regulating Zeb-1.

<p>(A) Cells were transfected with scramble, Snail or/and Zeb-1 siRNAs for 48 hours. E-cadherin, Zeb-1, and Snail protein levels were determined by Western blotting analysis. Actin was used as loading control. (B) E-cadherin expression was determined by quantitative real-time PCR analysis in control and 14-3-3ε overexpression cells. These data are from three independent experiments and presented as the mean ± SD. **<i>P</i><0.01. (C) Expression level and subcellular localization of E-cadherin was examined by immunofluorescent confocal microscopy. (D) 14-3-3ε siRNA dose-dependently decreased Zeb-1/Snail and restore E-cadherin expression in SK-Hep1 cells. Actin was used as loading control.</p

14-3-3ε promotes epithelial-mesenchymal transition.

<p>(A) Expressions of epithelial and mesenchymal markers included E-cadherin, N-cadherin, and vimentin in control and 14-3-3ε overexpression cells were determined by Western blot analysis and by (B) Immunofluorescent confocal microscopy. (C) 14-3-3ε siRNA-restored E-cadherin and -reduced N-cadherin as well as vimentin expression were determined by Western blotting analysis and by (D) Immunofluorescent confocal microscopy. Actin was used as loading control for protein determination.</p

Reversed correlation of 14-3-3ε with E-cadherin expression of hepatocellular carcinoma tumors.

<p>(A) Representative expression of 14-3-3ε and E-cadherin in primary tissue of HCC examined by immunohistochemical analysis. Original magnification, ×200. (B) Significantly reversed correlation of 14-3-3ε with E-cadherin expression in primary HCC tumors was analyzed by Chi-square test.</p

Kaplan-Meier analysis of 14-3-3ε and E-cadherin expression with prognostic outcomes in primary HCC tumors.

<p>E-cadherin positive expression reveals a (A) lower metastatic risk (<i>P</i> = 0.047), and (B) better overall survival rate (<i>P</i> = 0.013) when compared with E-cadherin negative in 14-3-3 positive patients.</p