Comparison of Microarray Platforms for Measuring Differential MicroRNA Expression in Paired Normal/Cancer Colon Tissues

Background

Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples.

Methodology/Principal Findings

We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport.

Conclusions

Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs – gene expression integration approach.     

Time of Progression to Osteopenia/Osteoporosis in Chronically HIV-Infected Patients: Screening DXA Scan

Background

Algorithms for bone mineral density (BMD) management in HIV-infected patients are lacking. Our objective was to assess how often a dual-energy x-ray absorptiometry (DXA) scan should be performed by assessing time of progression to osteopenia/osteoporosis.

Methods

All DXA scans performed between 2000 and 2009 from HIV-infected patients with at least two DXA were included. Time to an event (osteopenia and osteoporosis) was assessed using the Kaplan–Meier method. Strata (tertiles) were defined using baseline minimum T scores. Differences between strata in time to an event were compared with the log-rank test.

Results

Of 391 patients (1,639 DXAs), 49.6% had osteopenia and 21.7% osteoporosis at their first DXA scan. Of the 112 (28.6%) with normal BMD, 35.7% progressed to osteopenia; median progression time was 6.7 years. These patients were stratified: “low-risk" (baseline minimum T score >−0.2 SD), “middle-risk" (between −0.2 and −0.6 SD), and “high-risk" (from −0.6 to −1 SD); median progression time to osteopenia was 8.7, >7.2, and 1.7 years, respectively (p<0.0001). Of patients with osteopenia, 23.7% progressed to osteoporosis; median progression time was >8.5 years. Progression time was >8.2 years in “low-risk" tertile (T score between −1.1 and −1.6 SD), >8.5 years in “middle-risk" (between −1.6 and −2), and 3.2 years in “high-risk" (from −2 to −2.4) (p<0.0001).

Conclusions

Our results may help to define the BMD testing interval. The lowest T score tertiles would suggest recommending a subsequent DXA in 1–2 years; in the highest tertiles, ≥6 years. Early intervention in patients with bone demineralization could reduce fracture–related morbidity/mortality.     

Socioeconomic and nutritional factors account for the association of gastric cancer with amerindian ancestry in a latin american admixed population

Gastric cancer is one of the most lethal types of cancer and its incidence varies worldwide, with the Andean region of South America showing high incidence rates. We evaluated the genetic structure of the population from Lima (Peru) and performed a case-control genetic association study to test the contribution of African, European, or Native American ancestry to risk for gastric cancer, controlling for the effect of non-genetic factors. A wide set of socioeconomic, dietary, and clinic information was collected for each participant in the study and ancestry was estimated based on 103 ancestry informative markers. Although the urban population from Lima is usually considered as mestizo (i.e., admixed from Africans, Europeans, and Native Americans), we observed a high fraction of Native American ancestry (78.4% for the cases and 74.6% for the controls) and a very low African ancestry (<5%). We determined that higher Native American individual ancestry is associated with gastric cancer, but socioeconomic factors associated both with gastric cancer and Native American ethnicity account for this association. Therefore, the high incidence of gastric cancer in Peru does not seem to be related to susceptibility alleles common in this population. Instead, our result suggests a predominant role for ethnic-associated socioeconomic factors and disparities in access to health services. Since Native Americans are a neglected group in genomic studies, we suggest that the population from Lima and other large cities from Western South America with high Native American ancestry background may be convenient targets for epidemiological studies focused on this ethnic group.     

Obesity and postprandial lipemia in adolescents: risk factors for cardiovascular disease

In the last 50 years, obesity has become a global epidemic and is one of the main public health problems in many parts of the world. Adolescence is a critical period regarding weight control. The factors determining obesity include a complex group of interrelated biological, behavioral and environmental factors which reinforce each other. In children and adolescents, obesity is associated with premature cardiovascular diseases, diabetes mellitus type 2, acanthosis nigricans, respiratory and skeletal muscle problems, as well as psychological problems. The clinical manifestations of cardiovascular disease begin in middle age. Nevertheless, studies indicate that the atherosclerotic process begins to develop during childhood. Postprandial hyperlipemia is a physiological process that occurs several times a day after the complete absorption of a diet including lipids and has been suggested as a risk factor for coronary heart disease (CHD). New study areas include the effects of different fatty acids, lipid sources (endogenous and exogenous), and the effect ingesting alcoholic beverages during meals. Given the evidence that postprandial lipidemia is an independent risk factor for CHD, it is vital to establish normative values for children and adolescents such that more effective and efficient preventive and therapeutic measures can be adopted.     

Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

The Use of Ultrasonography to Study Teratogenicity in Ruminants: Evaluation of Ipomoea carnea in Goats

Background: Ipomoea carnea (I. carnea) is a poisonous plant found in Brazil and other tropical countries that often poison livestock. The plant contains the alkaloids calystegines and mainly swainsonine, which inhibit cellular enzymes and cause systematic cell death. The objective of this study was to evaluate the perinatal effects of I. carnea in goats. Methods: Forty‐seven pregnant goats were randomly allocated into 5 treatment groups and given the following doses (g/kg BW) of I. carnea: 0 (IC0), 1.0 (IC1), 3.0 (IC3), 5.0 (IC5) and 7.5 (IC7). The treatment animals were given fresh I. carnea from day 27 of gestation to parturition. Weight gains and serum biochemistry were evaluated. Fetuses were evaluated using ultrasonographic measurements. Results: Goats from the IC7 group showed clinical signs of poisoning. Ultrasound examination revealed that I. carnea feeding in all treatment groups reduced fetal movement compared to the controls. There was an increase in the total number of birth defects (retrognathia and arthrogyposis) in the IC7 and IC5 groups compared to the controls. Conclusion: The results show that I. carnea has teratogenic potential in goats. In addition, ultrasounds were useful in evaluating fetotoxicity and teratogenicity. Birth Defects Res (Part B) 00:1–7, 2012. © 2012 Wiley Periodicals, Inc.     

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

Phytochemical Profile and Nutraceutical Value of Old and Modern Common Wheat Cultivars

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems.Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases.     

Analysis of vaginal lactobacilli from healthy and infected Brazilian women

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H(2)O(2)-producing lactobacilli were not associated with protection against VVC.