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.     

AN ELECTRON MICROSCOPE STUDY OF DENERVATION ATROPHY IN RED AND WHITE SKELETAL MUSCLE FIBERS

A study, mainly by electron microscopy, has been made on two leg muscles of rat, in the course of atrophy experimentally induced by total denervation. As a preliminary the chief distinctive features of the soleus, chosen as a representative of pure red muscle, and of the gastrocnemius, representative of pure white muscle, are described. Two major phases of atrophy, somewhat overlapping in time, were observed. In the first, a degenerative autolytic process takes place in areas of the fiber, with loss of striation. It can be detected as early as the 7th day, but the maximum is observed at the 14th day, and accounts for a gross weight loss of 50 per cent. The first alteration appears in the Z lines; disorder in the disposition of filaments then follows. The process occurs very rapidly, leaving large areas in the cell in which one can detect only ground substance, glycogen, rare randomly disposed vesicular elements, and some mitochondria. Several lysosomes and masses of lipoproteins, which assume the configuration of concentric lamellae, show up in these fibers. Subsequently large parts of the waste sarcoplasm are discarded into the intercellular spaces. In the second major phase the so called "simple" atrophy takes place. The process starts early, but its effects are more detectable after 1 month. In this period, single myofibrils undergo different degrees of reduction in diameter, while the spatial disposition of primary and secondary filaments inside the fibrils remains normal. The appearance of the fibrils in longitudinal sections suggests that the process takes place by the detachment of filaments from the periphery of the fibrils and by their subsequent breakdown in the interfibrillary spaces. The sarcoplasmic reticulum is still well preserved, and relatively overdeveloped. Mitochondria disappear in parallel with the contractile material.     

Cobalamin and microbial plankton dynamics along a coastal to offshore transect in the Eastern North Atlantic Ocean

Cobalamin (B12) is an essential cofactor that is exclusively synthesized by some prokaryotes while many prokaryotes and eukaryotes require an external supply of B12. The spatial and temporal availability of B12 is poorly understood in marine ecosystems. Field measurements of B12 along with a large set of ancillary biotic and abiotic factors were obtained during three oceanographic cruises in the NW Iberian Peninsula, covering different spatial and temporal scales. B12 concentrations were remarkably low (<1.5 pM) in all samples, being significantly higher at the subsurface Eastern North Atlantic Central Water than at shallower depths, suggesting that B12 supply in this water mass is greater than demand. Multiple regression models excluded B12 concentration as predictive variable for phytoplankton biomass or production, regardless of the presence of B12-requiring algae. Prokaryote production was the best predictor for primary production, and eukaryote community composition was better correlated with prokaryote community composition than with nutritional resources, suggesting that biotic interactions play a significant role in regulating microbial communities. Interestingly, co-occurrence network analyses based on 16S and 18S rRNA sequences allowed the identification of significant associations between potential B12 producers and consumers (e.g. Thaumarchaeota and Dynophyceae, or Amylibacter and Ostreococcus respectively), which can now be investigated using model systems in the laboratory.     

Death and renal transplantation among Aboriginal people undergoing dialysis

Background

Despite the increase in the number of Aboriginal people with end-stage renal disease around the world, little is known about their health outcomes when undergoing renal replacement therapy. We evaluated differences in survival and rate of renal transplantation among Aboriginal and white patients after initiation of dialysis.

Methods

Adult patients who were Aboriginal or white and who commenced dialysis in Alberta, Saskatchewan or Manitoba between Jan. 1, 1990, and Dec. 31, 2000, were recruited for the study and were followed until death, transplantation, loss to follow-up or the end of the study (Dec. 31, 2001). We used Cox proportional hazards models to examine the effect of race on patient survival and likelihood of transplant, with adjustment for potential confounders.

Results

Of the 4333 adults who commenced dialysis during the study period, 15.8% were Aboriginal and 72.4% were white. Unadjusted rates of death per 1000 patient-years during the study period were 158 (95% confidence interval [CI] 144–176) for Aboriginal patients and 146 (95% CI 139–153) for white patients. When follow-up was censored at the time of transplantation, the age-adjusted risk of death after initiation of dialysis was significantly higher among Aboriginal patients than among white patients (hazard ratio [HR] 1.15, 95% CI 1.02–1.30). The greater risk of death associated with Aboriginal race was no longer observed after adjustment for diabetes mellitus and other comorbid conditions (adjusted HR 0.89, 95% CI 0.77–1.02) and did not appear to be associated with socioeconomic status. During the study period, unadjusted transplantation rates per 1000 patient-years were 62 (95% CI 52–75) for Aboriginal patients and 133 (95% CI 125–142) for white patients. Aboriginal patients were significantly less likely to receive a renal transplant after commencing dialysis, even after adjustment for potential confounders (HR 0.43, 95% CI 0.35–0.53). In an additional analysis that included follow-up after transplantation for those who received renal allografts, the age-adjusted risk of death associated with Aboriginal race (HR 1.36, 95% CI 1.21–1.52) was higher than when follow-up after transplantation was not considered, perhaps because of the lower rate of transplantation among Aboriginals.

Interpretation

Survival among dialysis patients was similar for Aboriginal and white patients after adjustment for comorbidity. However, despite universal access to health care, Aboriginal people had a significantly lower rate of renal transplantation, which might have adversely affected their survival when receiving renal replacement therapy.In North America and the Antipodes, the incidence of diabetes among adolescent and adult Aboriginals has risen dramatically,^1,2,3,4 with corresponding increases in the prevalence of diabetic nephropathy.^5,6,7 Aboriginal people in Canada have experienced disproportionately high incidence rates of end-stage renal disease (ESRD), with an 8-fold increase in the number of prevalent dialysis patients between 1980 and 2000.⁸ Although the incidence of ESRD appears to have decreased in recent years, the prevalence of diabetes mellitus and its complications are rising, especially among young people.^9,10,11Most work evaluating health outcomes among Aboriginal people considers either the general population¹²or diseases for which interventions are implemented over a short period, such as alcohol abuse,¹³ injury¹⁴ or critical illness.¹⁵ Death and markers of poor health are significantly more common among Aboriginal people than among North Americans of European ancestry, perhaps because of the greater prevalence of diabetes mellitus, adverse health effects due to lower socioeconomic status¹⁶ and reduced access to primary care.¹⁷ Aboriginal patients may also face unique barriers to care, including mistrust of non-Aboriginal providers, institutional discrimination or preference for traditional remedies.¹⁸ These factors may be most relevant when contact with physicians is infrequent, which obstructs development of a therapeutic relationship. In contrast, ESRD is a chronic illness that requires ongoing care from a relatively small, stable multidisciplinary team.Although recent evidence highlights racial inequalities in morbidity and mortality among North Americans with ESRD, most studies have focused on black or Hispanic populations.¹⁹We conducted this study to evaluate rates of death and renal transplantation among Aboriginal people after initiation of dialysis in Alberta, Saskatchewan and Manitoba.     

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.     

Twin seasonality in a rural Catalonian population

The seasonality of twinning in the Spanish populations has not been studied until now. Differences between seasonal distribution of the twin conceptions and those of the single births have been observed in other populations. The aim of this work is to explore the frequency of twinning in a rural population from Catalonia during the nineteenth century, as well as the seasonality patterns characterizing each of the twinning types. Data corresponding to all births recorded at Tortosa (South Catalonia) from 1801 to 1900 have been analyzed in order to study the twinning distribution. The distribution of the moving averages of the monthly rates of twins shows a peak in autumn. Twinning distribution differs from the total births' distribution in Tortosa. This fact is very clear in the case of unlike-sexed twins that have their greater incidence in the last quarter of the year, while the total maternities have their peak in the first one.     

Development of a new class of potential antiatherosclerosis agents: NO-donor antioxidants

A new class of NO-donor phenol derivatives is described. The products were obtained by joining appropriate phenols with either nitrooxy or 3-phenylsulfonylfuroxan-4-yloxy moieties. All the compounds proved to inhibit the ferrous salt/ascorbate induced lipidic peroxidation of membrane lipids of rat hepatocytes. They were also capable of dilating rat aorta strips precontracted with phenylephrine.     

Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats

Background  

In the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy.     

Effects on interfacial properties and cell adhesion of surface modification by pectic hairy regions

Polystyrene Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of two different enzymatically modified hairy regions (HRs) from pectin containing, for example, rhamnogalacturonan-I and xylogalacturonan structural elements. The two polysaccharide preparations share the same structural elements of apple pectin, but the relative amounts and lengths of the neutral side chains present differ. Surface analysis by X-ray photoelectron spectroscopy, contact angle measurement, and atomic force microscope (AFM) force-separation curves was used to characterize the effects on surface chemistry and interfacial forces of the surface modification process. Cell adhesion experiments using continuous L-929 fibroblasts and primary aortic smooth muscle cells were performed to evaluate the effect of the polysaccharide nature on cell adhesion. Results show that immobilization of the HR affects the interfacial field of forces and the cell behavior: "equilibrium" contact angles, obtained by a recently introduced vibrational approach, decrease after HR immobilization reaching a value close to 20 degrees . AFM force-separation curves show a more extended (or softer) interface in the case of the HR bearing longer side chains. Accordingly, depending on the HR preparation, cells shifted from spread morphology and adhesion behavior quantitatively comparable to that observed on conventional tissue culture polystyrene to rounded morphology and significantly lower adhesion. These data show that engineering of plant pectins can be a valuable tool to prepare novel and finely tuned polysaccharides having different chemico-physical and biological properties, to be used in the surface modification of medical devices and materials.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.