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.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

The WHO Maternal Near-Miss Approach and the Maternal Severity Index Model (MSI): Tools for Assessing the Management of Severe Maternal Morbidity

Objectives

To validate the WHO maternal near-miss criteria and develop a benchmark tool for severe maternal morbidity assessments.

Methods

In a multicenter cross-sectional study implemented in 27 referral maternity hospitals in Brazil, a one-year prospective surveillance on severe maternal morbidity and data collection was carried out. Diagnostic accuracy tests were used to assess the validity of the WHO maternal near-miss criteria. Binary logistic regression was used to model the death probability among women with severe maternal complications and benchmark the management of severe maternal morbidity.

Results

Of the 82,388 women having deliveries in the participating health facilities, 9,555 women presented pregnancy-related complications, including 140 maternal deaths and 770 maternal near misses. The WHO maternal near-miss criteria were found to be accurate and highly associated with maternal deaths (Positive likelihood ratio 106.8 (95% CI 99.56–114.6)). The maternal severity index (MSI) model was developed and found to able to describe the relationship between life-threatening conditions and mortality (Area under the ROC curve: 0.951 (95% CI 0.909–0.993)).

Conclusion

The identification of maternal near-miss cases using the WHO list of pregnancy-related life-threatening conditions was validated. The MSI model can be used as a tool for benchmarking the performance of health services managing women with severe maternal complications and provide case-mix adjustment.     

Fixed Differences in the paralytic Gene Define Two Lineages within the Lutzomyia longipalpis Complex Producing Different Types of Courtship Songs

The sand fly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae), the most important vector of American visceral leishmaniasis, is widely distributed in Latin America. There is currently a consensus that it represents a species complex, however, the number and distribution of the different siblings is still uncertain. Previous analyses have indicated that Brazilian populations of this vector can be divided into two main groups according to the type of courtship song (Burst vs. Pulse) males produce during copulation. Nevertheless, no diagnostic differences have been observed between these two groups with most molecular markers used to date. We analyzed the molecular divergence in a fragment of the paralytic (para) gene, a locus involved in the control of courtship songs in Drosophila, among a number of Lu. longipalpis populations from Brazil producing Burst and Pulse-type songs. Our results revealed a very high level of divergence and fixed differences between populations producing the two types of songs. We also compared Lu. longipalpis with a very closely related species, Lutzomyia cruzi, which produces Burst-type songs. The results indicated a higher number of fixed differences between Lu. cruzi and the Pulse-type populations of Lu. longipalpis than with those producing Burst-type songs. The data confirmed our previous assumptions that the presence of different sibling species of the Lu. longipalpis complex in Brazil can be divided into two main groups, one representing a single species and a second more heterogeneous group that probably represents a number of incipient species. We hypothesize that para might be one of the genes directly involved in the control of the courtship song differences between these two groups or that it is linked to other loci associated with reproductive isolation of the Brazilian species.     

Schwartz Formula: Is One k-Coefficient Adequate for All Children?

Background/Objective

Plasma-creatinine-based equations to estimate the glomerular filtration rate are recommended by several clinical guidelines. In 2009, Schwartz et al. adapted the traditional Schwartz equation to children and adolescents but did not find different k-coefficients between children and adolescents (k = 36.5 for all patients). We reevaluated the coefficient of the 2009-Schwartz formula according to sex and age in a pediatric population.

Patients/Methods

We used linear mixed-effects models to reestimate the 2009-Schwartz k-coefficient in 360 consecutive French subjects aged 1 to 18 years referred to a single centre between July 2003 and July 2010 (965 measurements). We assessed the agreement between the estimated glomerular filtration rate obtained with the new formula (called Schwartz-Lyon) and the rate measured by inulin clearance. We then compared this agreement to the one between the measured glomerular filtration rate and 2009-Schwartz formula, first in the French then in a Swedish cohort.

Results

In Schwartz-Lyon formula, k was estimated at 32.5 in boys <13 years and all girls and at 36.5 in boys aged ≥13 years. The performance of this formula was higher than that of 2009-Schwartz formula in children <13 years. This was first supported by a statistically significant reduction of the overestimation of the measured glomerular filtration rate in both cohorts, by better 10% and 30% accuracies, and by a better concordance correlation coefficient.

Conclusions

The performance and simplicity of Schwartz formula are strong arguments for its routine use in children and adolescents. The specific coefficient for children aged <13 years further improves this performance.     

Moniliophthora perniciosa Necrosis- and Ethylene-Inducing Protein 2 (MpNep2) as a Metastable Dimer in Solution: Structural and Functional Implications

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.     

Q-Elastography in the Presurgical Diagnosis of Thyroid Nodules with Indeterminate Cytology

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.     

Appraisal of WHO Guidelines in Maternal Health Using the AGREE II Assessment Tool

In 2007, the World Health Organization (WHO) received a criticism for a lack of transparency and systematic methods in the development of guidelines, which were at that time perceived as substantially driven by expert opinion. In this paper we assessed the quality of maternal and perinatal health guidelines developed since then. We used the Appraisal of Guidelines for Research and Evaluation (AGREE) II tool to evaluate the quality of methodological rigour and transparency of four different WHO guidelines published between 2007 and 2011. Our findings showed high scores among the most recent guidelines on maternal and perinatal health suggesting higher quality. However, there is still potential for improvement, especially in including different stakeholder views, transparency of guidelines regarding the role of the funding body and presentation of the guideline document.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.