Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells.

Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. Here, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process we refer to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.

γH2AX in the S Phase after UV Irradiation Corresponds to DNA Replication and Does Not Report on the Extent of DNA Damage.

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX, the phosphorylated form of the histone variant H2AX, in the S phase within an asynchronous population of cells. γH2AX is often considered a definitive marker of DNA damage inside a cell. In this report, we show that γH2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double-strand breaks or in the inhibition of global transcription. Instead, γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of γH2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage, but the response elicited, which peaks in the S phase upon UV irradiation.

Monitoring global changes in chromatin compaction states upon localized DNA damage with tools of fluorescence anisotropy.

In the eukaryotic nucleus, DNA, packaged in the form of chromatin, is subject to continuous damage. Chromatin has to be remodeled in order to repair such damage efficiently. But compact chromatin may also be more refractory to damage. Chromatin responses during DNA double-strand break (DSB) repair have been studied with biochemistry or as indirect readouts for the physical state of the chromatin at the site of damage. Direct measures of global chromatin compaction upon damage are lacking. We used fluorescence anisotropy imaging of histone H2B-EGFP to interrogate global chromatin compaction changes in response to localized DSBs directly. Anisotropy maps were preserved in fixation and reported on underlying chromatin compaction states. Laser-induced clustered DSBs led to global compaction of even the undamaged chromatin. Live-cell dynamics could be coupled with fixed-cell assays. Repair factors, PARP1 and PCNA, were immediately recruited to the site of damage, though the local enrichment of PCNA persisted longer than that of PARP1. Subsequently, nodes of PCNA that incorporated deoxynucleotide analogs were observed in regions of low-anisotropy open chromatin, even away from the site of damage. Such fluorescence anisotropy-based readout of chromatin compaction may be used in the investigation of different forms of DNA damage.

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.

Association between PDE4D gene and ischemic stroke: recent advancements.

Stroke is a severe complication and a leading cause of death worldwide and genetic studies among different ethnicities has provided the basis for involvement of phosphodiesterase 4D (PDE4D) gene in cerebrovascular diseases. Recent advancements have evaluated the role of this gene in stroke and these studies have provided a stronger support for the involvement of this gene in stroke development and few studies also suggest that it may influence outcome. Furthermore, case-control studies and meta-analysis studies have provided strong evidence for certain variants in PDE4D to predispose to stroke only among certain ethnicities. Thus, this review focuses on recent progress made in PDE4D gene research involving genetic, molecular and pharmacological aspect. A strong conclusion has emerged that clearly indicates a pivotal role played by this gene in ischemic stroke globally. Studies have also noticeably highlighted that PDE4D gene/pathway can be a suitable drug target for managing stroke; however, a more comprehensive research is still required to understand the molecular and cellular intricacies this gene plays in stroke development, progression and its outcome.

Association of ACE gene I/D polymorphism and ACE levels with hemorrhagic stroke: comparison with ischemic stroke.

In the present study, we investigated the association of insertion/deletion polymorphism of ACE gene with genetic predisposition to hemorrhagic stroke and also determined the mean ACE activity levels in ischemic and hemorrhagic stroke patients. Two hundred hemorrhagic stroke, 200 ischemic stroke patients and 200 gender and age matched controls were recruited for the study. We found statistically significant difference in the genotypic distribution between hemorrhagic patients and controls for dominant, co-dominant and recessive models. Significant difference was observed in the allelic frequencies between hemorrhagic patients and controls. Multiple logistic regression analysis confirmed these findings [adjusted OR for DD genotype was 2.46 (95 % CI 1.43-4.21) and p = 0.001] and [adjusted OR for ID genotype was 5.45 (95 % CI 2.6-10.4) and p = 0.001]. We have already established the association of this polymorphism in ischemic stroke patients. Comparing hemorrhagic with ischemic stroke, we found a significant difference in genotypic distribution between the two [for II vs. DD, χ (2) = 4.75; p = 0.03, OR = 0.5 (95 % CI 0.27-0.93) and for DD vs. ID, χ (2) = 5.1; p = 0.02, OR = 1.8 (95 % CI 1.1-3.3)]. Our results indicate that DD genotype and D allele are important risk factors for the development of stroke. Individuals harboring DD genotype of ACE I/D polymorphism are more predisposed to hemorrhagic stroke than ischemic stroke. Further, the mean ACE activity level was found to be significantly higher in hemorrhagic and ischemic stroke in comparison with controls, but there was no significant difference in the levels found between the two types of stroke.

Association of -1382A>G CCL11 gene variant with ischemic stroke, its subtypes and hemorrhagic stroke in a South Indian population.

BACKGROUND: CCL11 (Eotaxin-1) is an important inflammatory cytokine belonging to the CC family of chemokines associated with a number of infection or inflammation-related diseases such as atherosclerosis and stroke. We investigated the association of CCL11 gene polymorphism rs4795895-1382A>G with ischemic and hemorrhagic stroke. MATERIALS AND METHODS: Six hundred and twenty ischemic stroke patients, 620 age- and sex-matched healthy controls, and 220 hemorrhagic stroke patients, 220 age- and sex-matched healthy controls were included in the present study. The CCL11 gene polymorphism rs4795895-1382A>G was determined using PCR-RFLP technique. RESULTS: We found a statistically significant difference in the genotypic distribution between ischemic stroke patients and controls (For GG vs. AA, χ² = 7.604; P < 0.001, Odds ratio = 2.793; 95% CI = 1.308-5.9). For GG vs. AA + AG, χ² = 44.8, P < 0.001, Odds ratio = 2.382 (95% CI = 1.842-3.081). A significant difference was observed in the frequency of G and A alleles in patients and controls (For G vs. A, χ² = 43.26; P < 0.001, Odds ratio = 2.127; 95% CI = 1.693-2.672). Statistically significant difference was observed in the genotypic distribution between hemorrhagic stroke patients and controls (For GG vs. AG, χ² = 26.78; P = 0.001, Odds ratio = 3.5; 95% CI = 2.162-5.824). A significant difference was observed in the frequency of G and A alleles in patients and controls (For G vs. A, χ² = 41.98; P = 0.001, Odds ratio = 4.1; 95% CI = 2.61-6.44). CONCLUSION: The results of the present study show that the GG genotype is a significant risk factor for ischemic as well as hemorrhagic stroke. Further, the frequency of the GG genotype was observed to be higher in hemorrhagic stroke patients in comparison with ischemic stroke. Evaluating the association with ischemic stroke subtypes, a significant association was observed with intracranial large artery atherosclerosis and lacunar stroke.

E-selectin gene (S128R) polymorphism in hemorrhagic stroke: comparison with ischemic stroke.

Increasing evidence suggests that genetic variation in inflammatory genes plays a pivotal role in pathogenesis of stroke. The aim of the present study was to evaluate the association of E-selectin S128R polymorphism with hemorrhagic stroke and also to evaluate the genotypic and allelic variation with ischemic stroke in a South Indian population from Andhra Pradesh. In this study, we recruited 250 hemorrhagic stroke patients along with 250 age and sex matched controls. The genotypes were determined using PCR-RFLP method and the strength of association between genotypes and hemorrhagic stroke was determined by odds ratio with 95% confidence interval (CI) and chi-square analysis. Allelic and genotypic frequencies of the polymorphism differed significantly between hemorrhagic stroke patients and controls (p<0.001). Significant association was also found following dominant (p<0.001) and co-dominant (p<0.001) models. On comparing the genotypic and allelic frequencies between ischemic and hemorrhagic stroke significant difference was found between the two stroke types (p<0.001). In conclusion, we found the AC genotype to be a significant risk factor for hemorrhagic stroke and we also found significant differences in AC genotype and C allele among the two stroke types. The genotypic and allelic variation between the ischemic and hemorrhagic stroke, suggests that E-selectin S128R mediated amplification of leukocytes onto endothelial cells, leading to secondary damage of brain cells is more pronounced in hemorrhagic stroke.

CRP gene (1059G>C) polymorphism and its plasma levels in ischemic stroke and hemorrhagic stroke in a south Indian population.

In the present study, we evaluated the association of 1059G>C polymorphism in C-reactive protein (CRP) gene with the risk of ischemic and hemorrhagic strokes. We did not find a significant association of this polymorphism with stroke. However, 2 % of mutants were observed in hemorrhagic stroke patients with a 0.01 frequency for the C allele. We also estimated the high-sensitivity C-reactive protein (hsCRP) levels in hemorrhagic stroke and compared the levels with our already published data on ischemic stroke. The hsCRP level in hemorrhagic stroke was found to be significantly elevated in comparison with that in controls (p<0.001). However, there was no difference in the mean value of hsCRP levels between types of stroke. In conclusion, the G>C polymorphism in the promoter region of the CRP gene is not abundant in the population and cannot be connected with different hsCRP levels and stroke prediction. The CRP level is a useful marker in stroke, but cannot help in differentiating between types of stroke.

Association of E-selectin gene polymorphism (S128R) with ischemic stroke and stroke subtypes.

E-selectin is an important inflammatory cytokine involved in the pathogenesis of various diseases such as atherosclerosis and stroke. We investigated the association of E-selectin gene polymorphism (S128R) with ischemic stroke and its subtypes. We studied 610 patients with ischemic stroke and 610 age- and sex-matched healthy controls. The ischemic stroke was classified according to Trial of Org10172 in Acute Stroke Treatment (TOAST). E-selectin gene polymorphism (S128R) was determined by polymerase chain reaction-restriction fragment length polymorphism technique. We found statistically significant difference in the genotypic distribution between patients and controls (for AC vs. AA, χ(2) = 49.5; p < 0.001, odds ratio = 5.47(95 % CI, 3.25-9.21). A significant difference was observed in the frequency of C and A alleles in patients and controls (for C vs. A, χ(2) = 47.4; p < 0.001, odds ratio = 5.13 (95 % CI, 3.06-8.57). Multiple logistic regression analysis revealed that the most predictive risk factor for stroke was AC genotype (adjusted odds ratio = 1.450 (95 % CI, 1.23-2.75) and p = 0.001), hypertension, smoking, and diabetes (p = 0.001 in each case). We also found a significant association of AC genotype with intracranial large artery atherosclerosis (p < 0.01, odds ratio = 9.37, (95 % CI, 5.31-16.5) and small artery occlusion (p < 0.0001, odds ratio = 9.81 (95 % CI, 4.94-19.4). Our results indicate that the individuals bearing AC genotype of E-selectin gene polymorphism (S128R) are more prone to stroke than AA genotype.

Association of SNP41, SNP56 and a novel SNP in PDE4D gene with stroke and its subtypes.

An association between phosphodiesterase 4D (PDE4D) gene and risk of stroke has been suggested by deCODE group in an Icelandic population. In the present case-control study we investigated the association of SNP41 (rs12153798) and SNP56 (rs702553) with ischemic stroke and stroke subtypes. Five hundred and sixteen ischemic stroke patients and 513 healthy age and sex matched controls were included in the study. The genotypes were determined by subjecting the PCR products to sequencing. Both the SNPs 56 and 41 associated significantly with stroke [adjusted OR=1.97; 95% CI (1.262-3.082); p=0.003: adjusted OR=5.42; 95% CI (3.45-8.5); p<0.001 respectively]. In addition to this, a novel SNP at position 59736747 T>G was found while sequencing the PCR products including SNP56. This novel SNP was found in patients as well as controls but did not show a significant association with the disease. We found significant association of SNPs 56 and 41 with large artery atherosclerosis, lacunar and cardioembolic stroke. In conclusion PDE4D gene plays a key part in the pathogenesis of ischemic stroke in the South Indian population from Andhra Pradesh.

Association of genetic variants of fibrinolytic system with stroke and stroke subtypes.

Genetic variants of tPA (PLAT) and PAI-1 genes have been suggested to be the risk factors for stroke. In the present case-control study we investigated the association of -7351C/T polymorphism (rs2020918) and I/D polymorphism of tPA gene and Insertion/deletion polymorphism (4G/5G) of PAI-1 gene with genetic predisposition to ischemic stroke. 516 stroke patients and 513, sex and age matched healthy controls were involved in the study. We did not find a significant association of tPA -7351C/T polymorphism and PAI-1 4G/5G polymorphism with stroke. However, in case of I/D polymorphism significant difference was observed in the genotypic distribution and allelic frequency between the stroke patients and healthy controls. DD genotype and D allele associated significantly with stroke (p=0.002 and <0.001 respectively). We also found significant association of I/D polymorphism with intracranial large artery atherosclerosis and stroke of undetermined etiology. Exploring the association between gene-gene interaction (26 combinations including the three variants) and stroke, we found that individuals with CC+4G4G+DD, CC+5G5G+ID, CT+4G5G+ID, CT+5G5G+II, CT+5G5G+ID and TT+4G5G+II had a significantly higher risk of stroke. The results of this study suggest that -7351C/T polymorphism of tPA and 4G/5G polymorphism of PAI-1 are not associated with stroke, while as DD genotype and D allele of tPA gene are important risk factors for ischemic stroke. Further we found that the subjects with different tPA and PAI genotype combinations displayed a significantly high risk for overall ischemic stroke suggesting that gene-gene interaction involving more variants may change the susceptibility of particular subjects to the disease.