A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells

Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.     

Unbiased segregation of fission yeast chromosome 2 strands to daughter cells

The base complementarity feature (Watson and Crick in Nature 171(4356):737–738, 1953) and the rule of semi-conservative mode of DNA replication (Messelson and Stahl in Proc Natl Acad Sci U S A 44:671–682, 1958) dictate that two identical replicas of the parental chromosome are produced during replication. In principle, the inherent strand sequence differences could generate nonequivalent daughter chromosome replicas if one of the two strands were epigenetically imprinted during replication to effect silencing/expression of developmentally important genes. Indeed, inheritance of such a strand- and site-specific imprint confers developmental asymmetry to fission yeast sister cells by a phenomenon called mating/cell-type switching. Curiously, location of DNA strands with respect to each other at the centromere is fixed, and as a result, their selected segregation to specific sister chromatid copies occurs in eukaryotic cells. The yeast system provides a unique opportunity to determine the significance of such biased strand distribution to sister chromatids. We determined whether the cylindrical-shaped yeast cell distributes the specific chromosomal strand to the same cellular pole in successive cycles of cell division. By observing the pattern of recurrent mating-type switching in progenies of individual cells by microscopic analyses, we found that chromosome 2 strands are distributed by the random mode in successive cell divisions. We also exploited unusual “hotspot” recombination features of this system to investigate whether there is selective segregation of strands such that oldest Watson-containing strands co-segregate in the diploid cell at mitosis. Our data suggests that chromosome 2 strands are segregated independently to those of the homologous chromosome.     

Retrospective comparative study of efficacy,safety and outcome of percutaneous intervention for Budd-Chiari syndrome patients with bilirubin less than 3 and 3–6 mg/dl

Objective:Comparing the efficacy, safety and outcome of percutaneous intrervention for Budd-Chiari Syndrome (BCS) patients with bilirubin less than 3 and 3–6 mg dl⁻¹.Methods and materials:188 BCS patients having serum bilirubin ≤6 mg dl⁻¹ and underwent percutaneous interventions were divided into two groups based on bilirubin level: 151 patients having bilirubin <3 mg dl⁻¹ were included in Group 1; and 37 patients having bilirubin 3–6 mg dl⁻¹ were included in Group 2. Both group were compare for technical success (successful recanalization of hepatic venous stenosis or creation of portocaval shunt with post-procedure gradient ≤5 mm of Hg), Safety (procedure-related mortality/morbidity or patient required transplantation) and outcome (resolution of clinical symptoms and survival).Results:Technical success was 94.7% in Group 1–89.1% in Group 2 with overall success rate was 93.6%. No significant differences observed between the two groups in regards to procedure related complication. Overall transplant-free survival at 1 and 5 years after intervention in both groups was 96.3 and 91.2% respectively. 1-year and 5-year survivals in Group 1 was 96.7%, and 93.1%, whereas Group 2 was 94.6 and 90.1% with no statically significantly difference between the two groups (p = 0.59). Percutaneous intervention results are good in patients having bilirubin up to 6 mg dl⁻¹, i.e. mild to moderate liver dysfunctions.Conclusion:Technical success, survival and outcome of percutaneous intervention in BCS patients having serum bilirubin 3–6 mg dl⁻¹ was comparable to patients having bilirubin level <3 mg dl⁻¹.Advances in knowledge:Percutaneous intervention treatment is suitable for treatment for symptomatic BCS patients having bilirubin up to 6 mg  dl⁻¹.     

Validation of the GRACE score for prognosis in Indian patients with acute coronary syndromes

AimTo validate the global registry of acute coronary events (GRACE) score in acute coronary syndromes (ACS) patients and study its angiographic correlation.Methods and resultsTwo-hundred and thirty-five ACS patients were studied for the combined endpoint of all-cause in-hospital mortality and non-fatal infarction/reinfarction. We tested the predictive accuracy of the composite GRACE score using the receiver operating characteristics (ROC) curve.Lower systolic blood pressure (SBP) (odds ratio [OR] 7.93, P=0.005), ST-segment deviation (OR 7.79, P=0.02) and cardiac biomarker positivity (OR > 6.52, P=0.01) were significantly associated with events. Serum creatinine > 1.4 mg/dL showed a trend towards statistical significance (OR 4.14, P=0.05), whereas age > 50 years (OR 3.62, P=not significant [NS]) and Killips class 4 (OR 2.71, P=NS) showed good association. The best value for predicting events was a GRACE score of > 217 and these patients were more likely to have double/triple vessel disease (P = 0.0009). The C statistic for the GRACE score was 0.75.ConclusionHigher GRACE score predicts in-hospital events and more severe angiographic coronary artery disease (CAD).     

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.     

Evaluation of oral health related to body mass index

Oral Diseases (2012) 18 , 748–755 Objective: Poor oral health has previously been related to high body mass index (BMI). We aimed at exploring the link between BMI and several oral health markers, after adjustment for dietary patterns and plasma insulin, both of which could act as mediators. Subjects and Methods: Dental examination was performed in a sample of 186 French subjects aged 35–64 years and selected from the general population to assess number of missing teeth, periodontitis, clinical attachment loss (CAL), probing pocket depth (PD), gingival index (GI) and plaque index (PI). Data collection also included a food‐frequency questionnaire. BMI (considered as outcome variable) was categorized into quartiles, and as BMI<25; 25 ≤BMI<30; and BMI ≥ 30 kg m^?2. Results: After adjustment for age, gender, education level, smoking, physical activity, energy intake and C‐reactive protein, BMI was statistically associated with missing teeth, PD and PI, but not with CAL, GI or periodontitis. After additional adjustment for ‘high‐carbohydrate’ diet and plasma insulin or HOMA (homeostasis model assessment) index for insulin resistance, the statistical relationship between BMI and oral variables remained significant only for PD and PI. Conclusions: Plaque index, reflecting dental plaque, and PD, closely linked with periodontal inflammation and infection, are statistically associated with high BMI and obesity, independently of dietary patterns and insulin resistance.