Mitochondrial oxidative stress elicits chromosomal instability after exposure to isocyanates in human kidney epithelial cells

The role of oxidative stress is often attributed in environmental renal diseases. Isocyanates, a ubiquitous chemical group with diverse industrial applications, are known to undergo bio-transformation reactions upon accidental and occupational exposure. This study delineates the role of isocyanate-mediated mitochondrial oxidative stress in eliciting chromosomal instability in cultured human kidney epithelial cells. Cells treated with 0.005 µM concentration of methyl isocyanate displayed morphological transformation and stress-induced senescence. Along the time course, an increase in DCF fluorescence indicative of oxidative stress, depletion of superoxide dismutase (SOD) and glutathione reductase (GR) and consistent accumulation of 8-oxo-dG were noticed. Thus, endogenous oxidative stress resulted in aberrant expression of p53, p21, cyclin E and CDK2 proteins, suggestive of deregulated cell cycle, chromosomal aberrations, centromeric amplification, aneuploidy and genomic instability.     

On being “actionable”: clinical sequencing and the emerging contours of a regime of genomic medicine in oncology

This article explores commercial, academic, and national initiatives aimed at using sequencing technologies to generate “actionable” genomic results that can be applied to the clinical management of oncology patients. We argue that the term “actionable” is not merely a buzzword, but signals the emergence of a distinctive sociotechnical regime of genomic medicine in oncology. Unlike other regimes of genomic medicine that are organized around assessing and managing inherited risk for developing cancer (e.g. BRCA testing), actionable regimes aim to generate predictive relationships between genetic information and drug therapies, thereby generating new kinds of clinical actions. We explore how these genomic results are made actionable by articulating them with existing clinical routines, clinical trials, regulatory regimes, and health care systems; and in turn, how clinical sequencing programs have begun to reconfigure knowledge and practices in oncology. Actionability regimes confirm the emergence of bio-clinical decision-making in oncology, whereby the articulation of molecular hypotheses and experimental therapeutics become central to patient care.     

Fetal mesenchymal stromal cells from cryopreserved human chorionic villi: cytogenetic and molecular analysis of genome stability in long-term cultures

Background aimsFirst-trimester chorionic villi (CV) are an attractive source of human mesenchymal stromal cells (hMSC) for possible applications in cellular therapy and regenerative medicine. Human MSC from CV were monitored for genetic stability in long-term cultures.MethodsWe set up a good manufacturing practice cryopreservation procedure for small amounts of native CV samples. After isolation, hMSC were in vitro cultured and analyzed for biological end points. Genome stability at different passages of expansion was explored by karyotype, genome-wide array-comparative genomic hybridization and microsatellite genotyping.ResultsGrowth curve analysis revealed a high proliferative potential of CV-derived cells. Immunophenotyping showed expression of typical MSC markers and absence of hematopoietic markers. Analysis of multilineage potential demonstrated efficient differentiation into adipocytes, osteocytes, chondrocytes and induction of neuro-glial commitment. In angiogenic experiments, differentiation in endothelial cells was detected by in vitro Matrigel assay after vascular endothelial growth factor stimulation. Data obtained from karyotyping, array-comparative genomic hybridization and microsatellite genotyping comparing early with late DNA passages did not show any genomic variation at least up to passage 10. Aneuploid clones appeared in four of 14 cases at latest passages, immediately before culture growth arrest.ConclusionsOur findings indicate that hCV-MSC are genetically stable in long-term cultures at least up to passage 10 and that it is possible to achieve clinically relevant amounts of hCV-MSC even after few stages of expansion. Genome abnormalities at higher passages can occasionally occur and are always associated with spontaneous growth arrest. Under these circumstances, hCV-MSC could be suitable for therapeutic purposes.     

Genomics reveals new landscapes for crop improvement

The sequencing of large and complex genomes of crop species, facilitated by new sequencing technologies and bioinformatic approaches, has provided new opportunities for crop improvement. Current challenges include understanding how genetic variation translates into phenotypic performance in the field.     

Rho-GTPase signaling drives melanoma cell plasticity

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.     

A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering

The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.     

Cubic regression-based degree of correction predicts the performance of whole bisulfitome amplified DNA methylation analysis

Epigenetic mechanisms, including DNA methylation, are important determinants in development and disease. There is a need for technologies capable of detecting small variations in methylation levels in an accurate and reproducible manner, even if only limited amounts of DNA are available (which is the case in many studies in humans). Quantitative methylation analysis of minute DNA amounts after whole bisulfitome amplification (qMAMBA) has been proposed as an alternative, but this technique has not been adequately standardized and no comparative study against conventional methods has been performed, that includes a wide range of methylation percentages and different target assays. We designed an experiment to compare the performance of qMAMBA and bisulfite-treated genomic (non-amplified) DNA pyrosequencing. Reactions were performed in duplicate for each technique in eight different target genes, using nine artificially constructed DNA samples with methylation levels ranging between 0% and 100% with intervals of 12.5%. Cubic polynomial curves were plotted from the experimental results and the real methylation values and the resulting equation was used to estimate new corrected data points. The use of the cubic regression-based correction benefits the accuracy and the power of discrimination in methylation studies. Additionally, dispersion of the new estimated data around a y = x line (R²) served to fix a cutoff that can discriminate, with a single 9-point curve experiment, whether whole bisulfitome amplification and subsequent qMAMBA can produce accurate methylation results. Finally, even with an optimized reagent kit, DNA samples subjected to whole bisulfitome amplification enhance the preferential amplification of unmethylated alleles, and subtle changes in methylation levels cannot be detected confidently.     

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.     

番茄褪绿病毒TaqMan荧光定量PCR快速检测方法的建立与应用

【目的】本研究旨在建立TaqMan实时荧光定量PCR(TaqMan RT-qPCR)技术,快速检测单头烟粉虱Bemisia tabaci体内的番茄褪绿病毒(tomato chlorosis virus,ToCV)。【方法】根据ToCV外壳蛋白保守序列设计了1对特异性引物和1条TaqMan探针,建立了TaqMan RT-qPCR方法;与常规PCR检测进行比较,检测该方法的灵敏度与特异性;并应用该方法对单头烟粉虱成虫体内ToCV进行了快速检测。【结果】本研究构建的TaqMan RT-qPCR检测ToCV的标准曲线,其循环阈值(Ct值)与模板浓度具有良好的线性关系,扩增效率为98%。该方法对ToCV的最低检测浓度为8.3×10 copies/μL,灵敏度是常规RT-PCR的1000倍。该方法与田间番茄两种重要病毒番茄黄化曲叶病毒(tomato yellow leaf curl virus,TYLCV)和番茄斑萎病毒(tomato spotted wilt virus,TSWV)检测无交叉反应。单头烟粉虱成虫ToCV检测结果表明,温室内ToCV侵染植株上烟粉虱携毒率为100%,田间烟粉虱的携毒率为30%。【结论】本研究建立的TaqMan RT-qPCR检测方法,可快速有效检测单头烟粉虱体内ToCV携毒情况,为该病毒病的防控提供了技术支撑。