Phenotyping Type 2 Diabetes in Terms of Myocardial Insulin Resistance and Its Potential Cardiovascular Consequences: A New Strategy Based on 18F-FDG PET/CT.

BACKGROUND: Systemic insulin resistance is generally postulated as an independent risk factor of cardiovascular events in type 2 diabetes (T2D). However, the role of myocardial insulin resistance (mIR) remains to be clarified. METHODS: Two 18F-FDG PET/CT scans were performed on forty-three T2D patients at baseline and after hyperinsulinemic-euglycemic clamp (HEC). Myocardial insulin sensitivity (mIS) was determined by measuring the increment in myocardial 18F-FDG uptake after HEC. Coronary artery calcium scoring (CACs) and myocardial radiodensity (mRD) were assessed by CT. RESULTS: After HEC, seventeen patients exhibited a strikingly enhancement of myocardial 18F-FDG uptake and twenty-six a marginal increase, thus revealing mIS and mIR, respectively. Patients with mIR showed higher mRD (HU: 38.95 [33.81-44.06] vs. 30.82 [21.48-38.02]; p = 0.03) and CACs > 400 (AU: 52% vs. 29%; p = 0.002) than patients with mIS. In addition, HOMA-IR and mIS only showed a correlation in those patients with mIR. CONCLUSIONS: 18F-FDG PET combined with HEC is a reliable method for identifying patients with mIR. This subgroup of patients was found to be specifically at high risk of developing cardiovascular events and showed myocardial structural changes. Moreover, the gold-standard HOMA-IR index was only associated with mIR in this subgroup of patients. Our results open up a new avenue for stratifying patients with cardiovascular risk in T2D.

IgCaller for reconstructing immunoglobulin gene rearrangements and oncogenic translocations from whole-genome sequencing in lymphoid neoplasms.

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.

A Translational In Vivo and In Vitro Metabolomic Study Reveals Altered Metabolic Pathways in Red Blood Cells of Type 2 Diabetes.

Clinical parameters used in type 2 diabetes mellitus (T2D) diagnosis and monitoring such as glycosylated haemoglobin (HbA1c) are often unable to capture important information related to diabetic control and chronic complications. In order to search for additional biomarkers, we performed a pilot study comparing T2D patients with healthy controls matched by age, gender, and weight. By using 1H-nuclear magnetic resonance (NMR) based metabolomics profiling of red blood cells (RBCs), we found that the metabolic signature of RBCs in T2D subjects differed significantly from non-diabetic controls. Affected metabolites included glutathione, 2,3-bisphophoglycerate, inosinic acid, lactate, 6-phosphogluconate, creatine and adenosine triphosphate (ATP) and several amino acids such as leucine, glycine, alanine, lysine, aspartate, phenylalanine and tyrosine. These results were validated by an independent cohort of T2D and control patients. An analysis of the pathways in which these metabolites were involved showed that energetic and redox metabolism in RBCs were altered in T2D, as well as metabolites transported by RBCs. Taken together, our results revealed that the metabolic profile of RBCs can discriminate healthy controls from T2D patients. Further research is needed to determine whether metabolic fingerprint in RBC could be useful to complement the information obtained from HbA1c and glycemic variability as well as its potential role in the diabetes management.

Combining metabolic profiling of plasma and faeces as a fingerprint of insulin resistance in obesity.

BACKGROUND & AIMS: Insulin resistance (IR) is one of the main risk factor for type 2 diabetes mellitus (T2DM). Nevertheless, its underlying pathophysiology is not completely established because IR is triggered by a complex interconnection of numerous factors impairing metabolism, promoting metabolome changes. METHODS: We used a metabolomics approach to identify plasma and faecal metabolites related to IR and obesity. We explored a cohort of 44 subjects at baseline, with 30 of them followed two years thereafter in a longitudinal study after an hypocaloric diet in the obese subjects. RESULTS: In all individuals as a whole, 11 plasma metabolites positively associated with BMI (acetoacetate, creatinine, glycerol, glycerol of lipids, VLDL, fatty esters, myo-inositol, phenylalanine, threonine, tyrosine and valine) and one negatively (phosphocholine), with similar associations at baseline and follow-up. Four of these metabolites (myo-inositol, valine, acetoacetate and phosphocholine) remained significant within obese and non-obese groups. Thirteen faecal metabolites positively associated with BMI at baseline and one negatively (glutamine). However, these correlations did not remain significant at follow-up. The correlations were not always consistent at baseline and at follow-up and the metabolites that showed significant correlations were different for the obese group compared with the control group. The percent change in plasma Δethanolamine, Δglucose, Δuracil and Δhypoxanthine were positively associated with ΔBMI. The percent change in plasma Δphosphocholine and of faecal Δhydroxyphenylacetate, and Δ2-hydroxyphenylacetate were associated with ΔHOMA-IR in those patients that lost weight. Faecal branched chain amino acids (BCAAs) in faeces were associated with IR, following a similar pattern to that described for plasma BCAAs. Choline derivates had an opposite behaviour. CONCLUSIONS: The integration of plasma and faecal metabolites represents a valuable fingerprint that could help in the identification of patients at risk for IR and in the design of novel therapeutic strategies to prevent IR and the development of overt T2DM in the context of obesity. The results are coherent with diet having a much greater impact on faecal metabolomic profile than on plasma metabolome.

Identification of somatic gene mutations in penile squamous cell carcinoma.

There is a lack of studies on somatic gene mutations and cell signaling driving penile carcinogenesis. Our objective was to analyze somatic mutations in genes downstream of EGFR in penile squamous cell carcinomas, especially the mTOR and RAS/MAPK pathways. We retrospectively analyzed somatic mutations in 10 in situ and 65 invasive penile squamous cell carcinomas by using Sequenom's Mass Spectrometry iPlex Technology and Oncocarta v1.0 Panel. The DNA was extracted from FFPE blocks and we identified somatic missense mutations in three in situ tumors and in 19 invasive tumors, mostly in PIK3CA, KRAS, HRAS, NRAS, and PDGFA genes. Somatic mutations in the PIK3CA gene or RAS family genes were neither associated with tumor grade, stage or outcome, and were equally often identified in hrHPV positive and in hrHPV negative tumors that showed no p53 expression. Mutations in PIK3CA, KRAS, and HRAS are frequent in penile squamous cell carcinoma and likely play a role in the development of p53-negative tumors. Although the presence of these mutations does not seem to correlate with tumoral behavior or outcome, they could be biomarkers of treatment failure with anti-EGFR mAb in patients with penile squamous cell carcinoma.

Lkb1 loss promotes tumor progression of BRAF(V600E)-induced lung adenomas.

Aberrant activation of MAP kinase signaling pathway and loss of tumor suppressor LKB1 have been implicated in lung cancer development and progression. Although oncogenic KRAS mutations are frequent, BRAF mutations (BRAF(V600E)) are found in 3% of human non-small cell lung cancers. Contrary to KRAS mutant tumors, BRAF(V600E)-induced tumors are benign adenomas that fail to progess. Interestingly, loss of tumor supressor LKB1 coexists with KRAS oncogenic mutations and synergizes in tumor formation and progression, however, its cooperation with BRAF(V600E) oncogene is unknown. Our results describe a lung cell population in neonates mice where expression of BRAF(V600E) leads to lung adenoma development. Importantly, expression of BRAF(V600E) concomitant with the loss of only a single-copy of Lkb1, overcomes senencence-like features of BRAF(V600E)-mutant adenomas leading malignization to carcinomas. These results posit LKB1 haploinsufficiency as a risk factor for tumor progression of BRAF(V600E) mutated lung adenomas in human cancer patients.

Epithelial-mesenchymal transition markers and HER3 expression are predictors of elisidepsin treatment response in breast and pancreatic cancer cell lines.

Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734) is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT) and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1) and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.

ErbB3 expression predicts sensitivity to elisidepsin treatment: in vitro synergism with cisplatin, paclitaxel and gemcitabine in lung, breast and colon cancer cell lines.

Irvalec® (elisidepsin trifluoroacetate, PM02734) is a novel marine-derived cyclic peptide belonging to the Kahaladide family of compounds, currently in clinical trials with preliminary evidence of antitumor activity. Previous studies have shown a correlation between elisidepsin sensitivity and expression of the ErbB3 receptor in a panel of NSCLC cell lines. We have studied the effect of elisidepsin on the ErbB3 pathway, characterizing the expression of all members of the ErbB (HER) family of receptors and their main downstream signaling effectors, such as Akt and MAPK. Interestingly, we observed a downregulation of ErbB3 upon elisidepsin treatment that correlates with a reduction in the Akt phosphorylation levels in the most sensitive cell lines, whereas ErbB3 levels are not affected in the less sensitive ones. Also, we observed that the basal levels of ErbB3 protein expression show a significant correlation with cell viability response against elisidepsin treatment in 14 different cell lines. Furthermore, we analyzed the combination of elisidepsin with different chemotherapeutics agents, such as cisplatin, paclitaxel and gemcitabine, in a panel of different breast (MDA-MB-435, MDA-MB-231 and MCF7), lung (HOP62, DV90 and A549) and colorectal cancer cell lines (DLD1 and HT29). IC50 values for the different drugs were tested. We observed a synergistic effect in all cell lines tested with any chemotherapeutic agent. More importantly, the two in vitro elisidepsin-resistant cell lines (MDA-MB-231 and HOP62) presented a synergistic effect in combination with cisplatin and paclitaxel, respectively. These results provide a rationale for further development of these combinations in an ongoing clinical trial.

Evolution of cysteine patterns in the large extracellular loop of tetraspanins from animals, fungi, plants and single-celled eukaryotes.

By analyzing the evolution of cysteine patterns in the large extracellular loop (LEL) of tetraspanins across all eukaryotes, we report the following: (1) the origin of the cysteine-cysteine-glycine (CCG) motif in the common ancestor of unikonts (Animalia, fungi and amoebozoa); (2) tracing cysteine motifs on an eukaryotic phylogeny which includes protists, animals and plants match organismal evolution; (3) using this evolutionary approach we have determined some of the cysteines in these proteins that are involved in specific bonds in the LEL. Our study provides a framework to better understand tetraspanin formation, diversification and the evolutionary history of these important proteins.

Intron evolution: testing hypotheses of intron evolution using the phylogenomics of tetraspanins.

BACKGROUND: Although large scale informatics studies on introns can be useful in making broad inferences concerning patterns of intron gain and loss, more specific questions about intron evolution at a finer scale can be addressed using a gene family where structure and function are well known. Genome wide surveys of tetraspanins from a broad array of organisms with fully sequenced genomes are an excellent means to understand specifics of intron evolution. Our approach incorporated several new fully sequenced genomes that cover the major lineages of the animal kingdom as well as plants, protists and fungi. The analysis of exon/intron gene structure in such an evolutionary broad set of genomes allowed us to identify ancestral intron structure in tetraspanins throughout the eukaryotic tree of life. METHODOLOGY/PRINCIPAL FINDINGS: We performed a phylogenomic analysis of the intron/exon structure of the tetraspanin protein family. In addition, to the already characterized tetraspanin introns numbered 1 through 6 found in animals, three additional ancient, phase 0 introns we call 4a, 4b and 4c were found. These three novel introns in combination with the ancestral introns 1 to 6, define three basic tetraspanin gene structures which have been conserved throughout the animal kingdom. Our phylogenomic approach also allows the estimation of the time at which the introns of the 33 human tetraspanin paralogs appeared, which in many cases coincides with the concomitant acquisition of new introns. On the other hand, we observed that new introns (introns other than 1-6, 4a, b and c) were not randomly inserted into the tetraspanin gene structure. The region of tetraspanin genes corresponding to the small extracellular loop (SEL) accounts for only 10.5% of the total sequence length but had 46% of the new animal intron insertions. CONCLUSIONS/SIGNIFICANCE: Our results indicate that tests of intron evolution are strengthened by the phylogenomic approach with specific gene families like tetraspanins. These tests add to our understanding of genomic innovation coupled to major evolutionary divergence events, functional constraints and the timing of the appearance of evolutionary novelty.