Stochastic exit from mitosis in budding yeast: Model predictions and experimental observations

Unlike many mutants that are completely viable or inviable, the CLB2-dbΔ clb5Δ mutant of Saccharomyces cerevisiae is inviable in glucose but partially viable on slower growth media such as raffinose. On raffinose, the mutant cells can bud and divide but in each cycle there is a chance that a cell will fail to divide (telophase arrest), causing it to exit the cell cycle. This effect gives rise to a stochastic phenotype that cannot be explained by a deterministic model. We measure the interbud times of wild-type and mutant cells growing on raffinose and compute statistics and distributions to characterize the mutant''s behavior. We convert a detailed deterministic model of the budding yeast cell cycle to a stochastic model and determine the extent to which it captures the stochastic phenotype of the mutant strain. Predictions of the mathematical model are in reasonable agreement with our experimental data and suggest directions for improving the model. Ultimately, the ability to accurately model stochastic phenotypes may prove critical to understanding disease and therapeutic interventions in higher eukaryotes.Key words: stochastic phenotype, mitotic exit, non-genetic variability, cell cycle modeling, computational biology, stochastic modeling, deterministic modeling     

Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study

The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.     

Quantitative determination of allele frequency in pooled DNA by using sequencing method

Quantitative determination of the allele frequency of single-nucleotide polymorphism (SNP) in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present such a simple, accurate, and inexpensive method for quantitative determining the allele frequency in pooled DNA samples. Three steps of DNA pooling, PCR amplification and sequencing are involved in this assay. Although direct determination of the allele frequency from the two allele-specific fluorescence intensities is possible, correction for differential response of alleles is important. We explored the effect of differential response of alleles on test statistics and provide a solution to this problem based on heterozygous fluorescence intensities. We demonstrate the accuracy and reliability of this assay on pooled DNA samples with pre-determined allele frequencies from 7.1% to 53.9%. The accuracy of allele frequency measurements is high, with a correlation coefficient of r2 = 0.997 between measured and known frequencies. We believe that by providing a means for SNP genotyping up to hundreds of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies.     

Exploration of the binding of proton pump inhibitors to human P450 2C9 based on docking and molecular dynamics simulation

Human P450 protein CYP2C9 is one of the major drug-metabolizing isomers, contributing to the oxidation of 16% of the drugs currently in clinical use. To examine the interaction mechanisms between CYP2C9 and proton pump inhibitions (PPIs), we used molecular docking and molecular dynamics (MD) simulation methods to investigate the conformations and interactions around the binding sites of PPIs/CYPP2C9. Results from molecular docking and MD simulations demonstrate that nine PPIs adopt two different conformations (extended and U-bend structures) at the binding sites and position themselves far above the heme of 2C9. The presence of PPIs changes the secondary structures and residue flexibilities of 2C9. Interestingly, at the binding sites of all PPI–CYP2C9 complexes except for Lan/CYP2C9, there are hydrogen-bonding networks made of PPIs, water molecules, and some residues of 2C9. Moreover, there are strong hydrophobic interactions at all binding sites for PPIs/2C9, which indicate that electrostatic interactions and hydrophobic interactions appear to be important for stabilizing the binding sites of most PPIs/2C9. However, in the case of Lan/2C9, the hydrophobic interactions are more important than the electrostatic interactions for stabilizing the binding site. In addition, an interesting conformational conversion from extended to U-bend structures was observed for pantoprazole, which is attributed to an H-bond interaction in the binding pocket, an internal π–π stacking interaction, and an internal electrostatic interaction of pantoprazole.     

Fossilized biophotonic nanostructures reveal the original colors of 47-million-year-old moths

Structural colors are generated by scattering of light by variations in tissue nanostructure. They are widespread among animals and have been studied most extensively in butterflies and moths (Lepidoptera), which exhibit the widest diversity of photonic nanostructures, resultant colors, and visual effects of any extant organism. The evolution of structural coloration in lepidopterans, however, is poorly understood. Existing hypotheses based on phylogenetic and/or structural data are controversial and do not incorporate data from fossils. Here we report the first example of structurally colored scales in fossil lepidopterans; specimens are from the 47-million-year-old Messel oil shale (Germany). The preserved colors are generated by a multilayer reflector comprised of a stack of perforated laminae in the scale lumen; differently colored scales differ in their ultrastructure. The original colors were altered during fossilization but are reconstructed based upon preserved ultrastructural detail. The dorsal surface of the forewings was a yellow-green color that probably served as a dual-purpose defensive signal, i.e. aposematic during feeding and cryptic at rest. This visual signal was enhanced by suppression of iridescence (change in hue with viewing angle) achieved via two separate optical mechanisms: extensive perforation, and concave distortion, of the multilayer reflector. The fossils provide the first evidence, to our knowledge, for the function of structural color in fossils and demonstrate the feasibility of reconstructing color in non-metallic lepidopteran fossils. Plastic scale developmental processes and complex optical mechanisms for interspecific signaling had clearly evolved in lepidopterans by the mid-Eocene.     

White to brown fat phenotypic switch induced by genetic and environmental activation of a hypothalamic-adipocyte axis

Living in an enriched environment with complex physical and social stimulation leads to improved cognitive and metabolic health. In white fat, enrichment induced the upregulation of the brown fat cell fate determining gene Prdm16, brown fat-specific markers, and genes involved in thermogenesis and β-adrenergic signaling. Moreover, pockets of cells with prototypical brown fat morphology and high UCP1 levels were observed in the white fat of enriched mice associated with resistance to diet-induced obesity. Hypothalamic overexpression of BDNF reproduced the enrichment-associated activation of the brown fat gene program and lean phenotype. Inhibition of BDNF signaling by genetic knockout or dominant-negative trkB reversed this phenotype. Our genetic and pharmacologic data suggest a mechanism whereby induction of hypothalamic BDNF expression in response to environmental stimuli leads to selective sympathoneural modulation of white fat to induce "browning" and increased energy dissipation.     

Taxol,a microtubule stabilizer,prevents ischemic ventricular arrhythmias in rats

Microtubule integrity is important in cardio‐protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca²⁺]_i) and Ca²⁺ transients by using fluorometric determination, spectrophotometric assays and Fura‐2‐AM and Fluo‐3‐AM, respectively. The expression and activity of sarcoplasmic reticulum Ca²⁺‐ATPase (SERCA2a) was also examined using real‐time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide‐coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 μM) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 μM). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca²⁺]_i and preserved the amplitude and decay times of Ca²⁺ transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.     

V2O5 Nano‐Electrodes with High Power and Energy Densities for Thin Film Li‐Ion Batteries

Nanostructured V₂O₅ thin films have been prepared by means of cathodic deposition from an aqueous solution made from V₂O₅ and H₂O₂ directly on fluorine‐doped tin oxide coated (FTO) glasses followed by annealing at 500°C in air, and studied as film electrodes for lithium ion batteries. XPS results show that the as‐deposited films contained 15% V⁴⁺, however after annealing all the vanadium is oxidized to V⁵⁺. The crystallinity, surface morphology, and microstructures of the films have been investigated by means of XRD, SEM, and AFM. The V₂O₅ thin film electrodes show excellent electrochemical properties as cathodes for lithium ion intercalation: a high initial discharge capacity of 402 mA h g^?1 and 240 mA h g^?1 is retained after over 200 cycles with a discharging rate of 200 mA g^?1 (1.3 C). The specific energy density is calculated as 900 W h kg^?1 for the 1^st cycle and 723 W h kg^?1 for the 180^th cycle when the films are tested at 200 mA g^?1 (1.3 C). When discharge/charge is carried out at a high current density of 10.5 A g^?1 (70 C), the thin film electrodes retain a good discharge capacity of 120 mA h g^?1, and the specific power density is over 28 kW kg^?1.     

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.     

Expression and Biological Significance of Leptin,Leptin Receptor,VEGF, and CD34 in Colorectal Carcinoma

To investigate the expression and biological significance of Leptin, Leptin receptor, Vascular Endothelial Growth Factor (VEGF), and CD34 protein in colorectal carcinoma tissues. The expression of Leptin, Leptin receptor, VEGF, and CD34 was detected in 68 cases of colorectal carcinoma tissues, paired para-carcinoma tissues and normal colorectal tissues by Immunohistochemical SP Method. The results and related clinicopathological data were analyzed. The positive rate of Leptin, Leptin receptor, and VEGF was significantly higher in colorectal carcinoma tissues than that in paired para-carcinoma tissues and normal colorectal tissues. The expression of Leptin, Leptin receptor, and VEGF was correlated with grade of tumor differentiation, depth of bowel wall invasion, lymph node metastasis, Dukes stage, distant metastasis, and lympho/vascular tumor embolization. Microvessel density (MVD) value in colorectal carcinoma was significantly higher than that in para-carcinoma tissues and normal colorectal tissues, and the density in para-carcinoma tissues was higher than that in normal colorectal tissues. The expression of Leptin, Leptin receptor, VEGF, and MVD value in colorectal carcinoma was positively correlated. In conclusion, microvessel density value is an important index of the growth, invasion, and metastasis of colorectal carcinoma. The binding of Leptin and Leptin receptor promotes the proliferation of colorectal carcinoma cells. The synergy between Leptin and VEGF accelerates the angiogenesis in colorectal carcinoma and accelerates the invasion and metastasis of the tumor cells.