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

The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met⁺ Hepatocellular Carcinoma

<div><p>Background</p><p>c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.</p><p>Methods</p><p>We utilized the human MHCC97-H c-Met positive (c-Met⁺) HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells) were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.</p><p>Results</p><p>We have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth <i>in vivo</i>. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth <i>in vitro</i>. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, <i>in vitro</i> studies demonstrate that combination therapy with PHA665752 and Gefitinib (an EGFR inhibitor) significantly reduced cell viability and increased apoptosis compared with either PHA665752 or Gefitinib treatment alone.</p><p>Conclusion</p><p>c-Met inhibition monotherapy is not sufficient to eliminate c-Met⁺ HCC tumor growth. Inhibition of both c-Met and EGFR oncogenic pathways provides superior suppression of HCC tumor growth. Thus, combination of c-Met and EGFR inhibition may represent a superior therapeutic regimen for c-Met⁺ HCC.</p></div

Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

Abstract Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.</p

Combined inhibition of EGFR and c-Met in c-Met⁺ HCC leads to superior suppression of tumor growth than c-Met inhibitor alone in c-Met⁺ HCC.

<p>XTT cell viability assay 48 hours after treatment of A) MHCC97-H and B) SNU-449 cells treated with EGFR inhibitor gefitinib, c-Met inhibitor PHA665752 or both inhibitors. C) Apoptosis by flow cytometry of MHCC97-H cells treated with EGFR inhibitor gefitinib (10 μM), c-Met inhibitor PHA665752 (1 μM) or both. D) PARP cleavage by immunoblot 48 hours after treatment of MHCC97-H cells with EGFR inhibitor gefitinib (10 μM), c-Met inhibitor PHA665752 (1 μM) or both inhibitors.</p

EGFR is a compensatory, not concomitant survival pathway in c-Met⁺ HCC.

<p>A) Immunoblot of c-Met^- cell lines Huh7 and Hep3B and c-Met⁺ cell lines MHCC97-L and MHCC97-H 24 hours post EGF treatment (0, 50, or 100 ng/ml) for EGFR, Akt and Erk signaling pathway activation. B) XTT cell viability assay of c-Met^-cell line Huh7 treated with c-Met inhibitor PHA665752 (1 μM), EGFR inhibitor gefitinib (10 μM) or DMSO control 48 hours after treatment. C) XTT cell viability assay of c-Met⁺ MHCC97-H treated with c-Met inhibitor PHA665752 (1 μM), EGFR inhibitor gefitinib (10 μM) or DMSO control 48 hours after treatment. D) XTT cell viability assay of c-Met⁺ SNU-449 cell line treated with c-Met inhibitor PHA665752 (5 μM), EGFR inhibitor gefitinib (10 μM) or DMSO control for 48 hours. *statistically significant compared to DMSO control by Student t-test (p <0.05).</p

Transforming growth factor alpha (TGF-α), an EGFR ligand, is regulated by PI3K/Akt signaling downstream of c-Met and can act as a compensatory survival mechanism during c-Met blockade in c-Met⁺ HCC.

<p>A) TGF-α mRNA expression by qRT-PCR in MHCC97-H cells treated with c-Met inhibitor PHA665752 (1 μM), PI3K inhibitor LY290042 (25 μM), or Mek inhibitor PD98059 (50 μM) 48 hours after treatment. B) Cell viability by XTT assay of MHCC97-H cells treated with varying doses of TGF-α and/or c-Met inhibitor PHA665752 (1 μM) 48 hours after treatment. C) Immunoblot of MHCC97-H cells treated with combinations of TGF-α ng/ml), c-Met inhibitor PHA665752 (1 μM), or gefitinib (10 μM) for 48 hours. Immunoblot was performed for c-Met, p-c-Met, EGFR, p-EGFR, Akt, p-Akt, Erk1/2, p-Erk1/2, ErbB3, and cleaved PARP.</p

siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC.

<p>A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met⁺ HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post transfection. C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p <0.05) 1.4-fold or greater change in expression between c-Met shRNA and scrambled shRNA cell lines was considered differentially expressed.</p

Suppression of c-Met in c-Met⁺ HCC upregulates ErbB3 predominantly through the PI3K/Akt signaling arm.

<p>(A) EGFR, ErbB2, and ErbB3 mRNA by qRT-PCR and (B) ErbB3 protein expression by immunoblot in MHCC97-H cells treated with c-Met inhibitor PHA665752 (1 μM) 48 hours after treatment. (C) ErbB3 mRNA by qRT-PCR and (D) protein by immunoblot in MHCC97-H cells treated with c-Met inhibitor PHA665752 (1 μM), PI3K inhibitor LY290042 (25 μM), or Mek inhibitor PD98059 (50 μM) 48 hours after treatment.</p