Integration Analysis of Three Omics Data Using Penalized Regression Methods: An Application to Bladder Cancer

Omics data integration is becoming necessary to investigate the genomic mechanisms involved in complex diseases. During the integration process, many challenges arise such as data heterogeneity, the smaller number of individuals in comparison to the number of parameters, multicollinearity, and interpretation and validation of results due to their complexity and lack of knowledge about biological processes. To overcome some of these issues, innovative statistical approaches are being developed. In this work, we propose a permutation-based method to concomitantly assess significance and correct by multiple testing with the MaxT algorithm. This was applied with penalized regression methods (LASSO and ENET) when exploring relationships between common genetic variants, DNA methylation and gene expression measured in bladder tumor samples. The overall analysis flow consisted of three steps: (1) SNPs/CpGs were selected per each gene probe within 1Mb window upstream and downstream the gene; (2) LASSO and ENET were applied to assess the association between each expression probe and the selected SNPs/CpGs in three multivariable models (SNP, CPG, and Global models, the latter integrating SNPs and CPGs); and (3) the significance of each model was assessed using the permutation-based MaxT method. We identified 48 genes whose expression levels were significantly associated with both SNPs and CPGs. Importantly, 36 (75%) of them were replicated in an independent data set (TCGA) and the performance of the proposed method was checked with a simulation study. We further support our results with a biological interpretation based on an enrichment analysis. The approach we propose allows reducing computational time and is flexible and easy to implement when analyzing several types of omics data. Our results highlight the importance of integrating omics data by applying appropriate statistical strategies to discover new insights into the complex genetic mechanisms involved in disease conditions.     

DNA methylation abnormalities in congenital heart disease

Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.     

Detection of a novel stem cell probably involved in normal turnover of the lung airway epithelium

Regeneration of the lung airway epithelium after injury has been extensively studied. In contrast, analysis of its turnover in healthy adulthood has received little attention. In the classical view, this epithelium is maintained in the steady‐state by the infrequent proliferation of basal or Clara cells. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its expression has also been detected in other stem cells. Lungs from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in neutral‐buffered formalin and paraffin‐embedded. Nestin expression was examined by an immunohistochemical peroxidase‐based method. Nestin‐positive cells were detected in perivascular areas and in connective tissue that were in close proximity of the airway epithelium. Also, nestin‐positive cells were found among the cells lining the airway epithelium. These findings suggest that nestin‐positive stem cells circulate in the bloodstream, transmigrate through blood vessels and localize in the lung airway epithelium to participate in its turnover. We previously reported the existence of similar cells able to differentiate into lung chondrocytes. Thus, the stem cell reported here might be a bone marrow‐derived mesenchymal stem cell (BMDMSC) able to generate several types of lung tissues. In conclusion, our findings indicate that there exist a BMDMSC in healthy adulthood that participates in the turnover of the lung airway epithelium. These findings may improve our knowledge about the lung stem cell biology and also provide novel approaches to therapy for devastating pulmonary diseases.     

Implications for human adipose‐derived stem cells in plastic surgery

Adipose‐derived stem cells (ADSCs) are a subset of mesenchymal stem cells (MSCs) that possess many of the same regenerative properties as other MSCs. However, the ubiquitous presence of ADSCs and their ease of access in human tissue have led to a burgeoning field of research. The plastic surgeon is uniquely positioned to harness this technology because of the relative frequency in which they perform procedures such as liposuction and autologous fat grafting. This review examines the current landscape of ADSC isolation and identification, summarizes the current applications of ADSCs in the field of plastic surgery, discusses the risks associated with their use, current barriers to universal clinical translatability, and surveys the latest research which may help to overcome these obstacles.     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.     

A proteomic approach to obesity and type 2 diabetes

The incidence of obesity and type diabetes 2 has increased dramatically resulting in an increased interest in its biomedical relevance. However, the mechanisms that trigger the development of diabetes type 2 in obese patients remain largely unknown. Scientific, clinical and pharmaceutical communities are dedicating vast resources to unravel this issue by applying different omics tools. During the last decade, the advances in proteomic approaches and the Human Proteome Organization have opened and are opening a new door that may be helpful in the identification of patients at risk and to improve current therapies. Here, we briefly review some of the advances in our understanding of type 2 diabetes that have occurred through the application of proteomics. We also review, in detail, the current improvements in proteomic methodologies and new strategies that could be employed to further advance our understanding of this pathology. By applying these new proteomic advances, novel therapeutic and/or diagnostic protein targets will be discovered in the obesity/Type 2 diabetes area.     

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.     

Regularity of Cardiac Rhythm as a Marker of Sleepiness in Sleep Disordered Breathing

Aim

The present study aimed to analyse the autonomic nervous system activity using heart rate variability (HRV) to detect sleep disordered breathing (SDB) patients with and without excessive daytime sleepiness (EDS) before sleep onset.

Methods

Two groups of 20 patients with different levels of daytime sleepiness -sleepy group, SG; alert group, AG- were selected consecutively from a Maintenance of Wakefulness Test (MWT) and Multiple Sleep Latency Test (MSLT) research protocol. The first waking 3-min window of RR signal at the beginning of each nap test was considered for the analysis. HRV was measured with traditional linear measures and with time-frequency representations. Non-linear measures -correntropy, CORR; auto-mutual-information function, AMIF- were used to describe the regularity of the RR rhythm. Statistical analysis was performed with non-parametric tests.

Results

Non-linear dynamic of the RR rhythm was more regular in the SG than in the AG during the first wakefulness period of MSLT, but not during MWT. AMIF (in high-frequency and in Total band) and CORR (in Total band) yielded sensitivity > 70%, specificity >75% and an area under ROC curve > 0.80 in classifying SG and AG patients.

Conclusion

The regularity of the RR rhythm measured at the beginning of the MSLT could be used to detect SDB patients with and without EDS before the appearance of sleep onset.