Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

Lower Extremity Amputations in Persons with and without Diabetes in Italy: 2001–2010

Objective

To analyze hospitalization for lower extremity amputations (LEAs) and amputee rates in persons with and without diabetes in Italy.

Research Design and Methods

All patients with LEAs in the period 2001–2010 were identified analyzing the National Hospital Discharge Record database. For each year, amputee and hospitalization rates for LEAs were calculated either for persons with diabetes or without. Time trend for major and minor amputations were analysed.

Results

From 2001 to 2010 a mean annual number of 11,639 individuals underwent a lower extremity amputation: 58.6% had diabetes accounting for 60.7% of total hospitalizations. In 2010, the crude amputee rate for LEAs was 20.4 per 100,000 inhabitants: 247.2 for 100.000 persons with diabetes, and 8.6 for those without diabetes. Having diabetes was associated to an increased risk of amputation (Poisson estimated RR 10.9, 95%CI 9.4–12.8). Over the whole period, a progressive reduction of amputee rates was observed for major amputations either among persons with diabetes (−30.7%) or without diabetes (−12.5%), while the rates of minor amputations increased progressively (+22.4%) among people without diabetes and were nearly stable in people with diabetes (−4.6%). A greater number of minor amputations were performed among persons with than without diabetes: in 2010, the minor-to-major ratio among persons with diabetes (2.5) was more than twice than in those without diabetes (1.0).

Conclusions

The nationwide analyses confirm a progressive reduction of hospitalization and amputee rates for major LEAs, suggesting an earlier and more diffuse approach aimed at limb salvage.     

Plasma lipidome is independently associated with variability in metabolic syndrome in Mexican American families

Plasma lipidome is now increasingly recognized as a potentially important marker of chronic diseases, but the exact extent of its contribution to the interindividual phenotypic variability in family studies is unknown. Here, we used the rich data from the ongoing San Antonio Family Heart Study (SAFHS) and developed a novel statistical approach to quantify the independent and additive value of the plasma lipidome in explaining metabolic syndrome (MS) variability in Mexican American families recruited in the SAFHS. Our analytical approach included two preprocessing steps: principal components analysis of the high-resolution plasma lipidomics data and construction of a subject-subject lipidomic similarity matrix. We then used the Sequential Oligogenic Linkage Analysis Routines software to model the complex family relationships, lipidomic similarities, and other important covariates in a variance components framework. Our results suggested that even after accounting for the shared genetic influences, indicators of lipemic status (total serum cholesterol, TGs, and HDL cholesterol), and obesity, the plasma lipidome independently explained 22% of variability in the homeostatic model of assessment-insulin resistance trait and 16% to 22% variability in glucose, insulin, and waist circumference. Our results demonstrate that plasma lipidomic studies can additively contribute to an understanding of the interindividual variability in MS.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Partial characterization and detergent solubilization of the putative glutathione chemoreceptor from hydra.

Feeding behavior in hydra is initiated by the association of glutathione (GSH) with a putative external chemoreceptor. In the present study, the binding of [35S]GSH to hydra membranes has been characterized. Nondisplaceable [35S]GSH binding which compromised previous analyses [Grosvenor, W., Bellis, S., Kass-Simon, G., & Rhoads, D. (1992) Biochim. Biophys. Acta (in press)] was eliminated by treating membranes with an inhibitor of GSH metabolism, borate in combination with L-serine. The specific binding which was not inhibited by borate/serine demonstrated many of the characteristics expected of a ligand/receptor interaction. The binding was rapid, reversible, and saturable. A Scatchard analysis of saturation isotherms indicated a dissociation constant (KD) of 3.4 microM, a value which is in good agreement with concentrations of glutathione which are known to induce feeding behavior. Hydra membranes were detergent-solubilized with 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 100 mM KCl, and 10% glycerol. The soluble fraction contained 40% of the original saturable, reversible GSH binding activity. The KD for GSH binding to the solubilized preparation was estimated as 2.7 microM, a valuable which is not appreciably different from the KD for binding to intact membranes. The fidelity of GSH binding in the solubilized preparation suggests that this preparation will be useful in further characterization of the putative glutathione chemoreceptor.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.