An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA

<p>Abstract</p> <p>Background</p> <p>Validation of microarrays data by quantitative real-time PCR (qPCR) is often limited by the low amount of available RNA. This raised the possibility to perform validation experiments on the amplified amino allyl labeled RNA (AA-aRNA) leftover from microarrays. To test this possibility, we used an ongoing study of our laboratory aiming at identifying new biomarkers of graft rejection by the transcriptomic analysis of blood cells from brain-dead organ donors.</p> <p>Results</p> <p>qPCR for ACTB performed on AA-aRNA from 15 donors provided Cq values 8 cycles higher than when original RNA was used (P < 0.001), suggesting a strong inhibition of qPCR performed on AA-aRNA. When expression levels of 5 other genes were measured in AA-aRNA generated from a universal reference RNA, qPCR sensitivity and efficiency were decreased. This prevented the quantification of one low-abundant gene, which was readily quantified in un-amplified and un-labeled RNA. To overcome this limitation, we modified the reverse transcription (RT) protocol that generates cDNA from AA-aRNA as follows: addition of a denaturation step and 2-min incubation at room temperature to improve random primers annealing, a transcription initiation step to improve RT, and a final treatment with RNase H to degrade remaining RNA. Tested on universal reference AA-aRNA, these modifications provided a gain of 3.4 Cq (average from 5 genes, P < 0.001) and an increase of qPCR efficiency (from -1.96 to -2.88; P = 0.02). They also allowed for the detection of a low-abundant gene that was previously undetectable. Tested on AA-aRNA from 15 brain-dead organ donors, RT optimization provided a gain of 2.7 cycles (average from 7 genes, P = 0.004). Finally, qPCR results significantly correlated with microarrays.</p> <p>Conclusion</p> <p>We present here an optimized RT protocol for validation of microarrays by qPCR from AA-aRNA. This is particularly valuable in experiments where limited amount of RNA is available.</p

Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetateinduced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.This work was supported by the Fundação para a Ciência e Tecnologia (FCT) research project PTDC/BIA-BCM/69448/2006 and FCT PhD grants for SA (SFRH/BD/64695/2009) and CO (SFRH/BD/77449/2011). This work was also supported by FEDER through POFC—COMPETE, and by national funds from FCT through the project PEst-C/BIA/UI4050/2011

Pharmacokinetic and exploratory exposure-response analysis of pertuzumab in patients with operable HER2-positive early breast cancer in the APHINITY study

Purpose: To characterize the pharmacokinetics (PK) of, and perform an exploratory exposure–response (E–R) analysis for, pertuzumab in patients with HER2-positive early breast cancer (EBC) within the APHINITY study (NCT01358877, BIG 4–11/BO25126/TOC4939G). Methods: A previously developed pertuzumab two-compartment linear population pharmacokinetic (popPK) model was subjected to external validation to examine appropriateness for describing pertuzumab concentrations from the APHINITY study. Pharmacokinetic drug–drug interactions (DDIs) between pertuzumab, trastuzumab, and chemotherapy were assessed by comparing observed serum or plasma Cmax, Cmin, and AUClast geometric mean ratios with 90% CIs. Predictions of pertuzumab Cmax,ss, Cmin,ss, and AUCss were derived from individual parameter estimates and used in an exploratory E–R analysis. Results: Using data from 72 patients, based on goodness-of-fit, the popPK model was deemed appropriate for predictions of individual exposures for subsequent comparisons to historical data, assessment of DDIs, and E–R analyses. No evidence of DDIs for pertuzumab on trastuzumab, trastuzumab on pertuzumab, or pertuzumab on chemotherapy PK was observed. Analyses of differences in exposure between patients with and without invasive disease-free survival events did not indicate improved efficacy with increased exposure. Overall Grade ≥ 3 diarrhea prevalence was higher with pertuzumab versus placebo, but was not greater with increasing pertuzumab exposure. No apparent E–R relationship was suggested with respect to other grade ≥ 3 AEs. Conclusion: Overall, the limited available data from this exploratory study suggest that no dose adjustments are needed for pertuzumab when administered in combination with trastuzumab and an EBC chemotherapy regimen

Mycobacterium tuberculosis Induces an Atypical Cell Death Mode to Escape from Infected Macrophages

BACKGROUND: Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds approximately 20 per macrophage but the precise nature of this demise has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system. CONCLUSIONS/SIGNIFICANCE: Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis

The propeptide of yeast cathepsin D inhibits programmed necrosis

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast

A microarray study of MPP(+)-treated PC12 Cells: Mechanisms of toxicity (MOT) analysis using bioinformatics tools

BACKGROUND: This paper describes a microarray study including data quality control, data analysis and the analysis of the mechanism of toxicity (MOT) induced by 1-methyl-4-phenylpyridinium (MPP(+)) in a rat adrenal pheochromocytoma cell line (PC12 cells) using bioinformatics tools. MPP(+ )depletes dopamine content and elicits cell death in PC12 cells. However, the mechanism of MPP(+)-induced neurotoxicity is still unclear. RESULTS: In this study, Agilent rat oligo 22K microarrays were used to examine alterations in gene expression of PC12 cells after 500 μM MPP(+ )treatment. Relative gene expression of control and treated cells represented by spot intensities on the array chips was analyzed using bioinformatics tools. Raw data from each array were input into the NCTR ArrayTrack database, and normalized using a Lowess normalization method. Data quality was monitored in ArrayTrack. The means of the averaged log ratio of the paired samples were used to identify the fold changes of gene expression in PC12 cells after MPP(+ )treatment. Our data showed that 106 genes and ESTs (Expressed Sequence Tags) were changed 2-fold and above with MPP(+ )treatment; among these, 75 genes had gene symbols and 59 genes had known functions according to the Agilent gene Refguide and ArrayTrack-linked gene library. The mechanism of MPP(+)-induced toxicity in PC12 cells was analyzed based on their genes functions, biological process, pathways and previous published literatures. CONCLUSION: Multiple pathways were suggested to be involved in the mechanism of MPP(+)-induced toxicity, including oxidative stress, DNA and protein damage, cell cycling arrest, and apoptosis

Cell death during sepsis: integration of disintegration in the inflammatory response to overwhelming infection

Sepsis is a major health problem and a leading cause of death worldwide. In recent years, a crescendo of attention has been directed to the mechanisms of cell death that develop during this disease, since these are viewed as important contributors to the proinflammatory and anti-inflammatory responses associated with poor outcome. Here we discuss mechanisms of cell death evident severe bacterial infection and sepsis including necrosis, apoptosis, pyroptosis, and extracellular trap-associated neutrophil death, with a particular emphasis on lymphocyte apoptosis and its contribution to the immunosuppressed phenotype of late sepsis. Individual bacterial pathogens express virulence factors that modulate cell death pathways and influence the sepsis phenotype. A greater knowledge of cell death pathways in sepsis informs the potential for future therapies designed to ameliorate immune dysfunction in this syndrome