Emerging Strategies and Effective Prevention Measures for Investigating the Association Between Stroke and Sudden Cardiac Fatality.

Stroke-related cardiac death is a significant concern for patients with stroke and their healthcare providers. It is a complex and multifaceted condition that requires careful management of both modifiable and non-modifiable risk factors. This review provides an overview of the pathophysiology, risk factors, and prevention strategies for stroke-related cardiac death. The review highlights the importance of identifying and managing modifiable risk factors such as hypertension, diabetes, and lifestyle factors, as well as non-modifiable risk factors such as age and genetics. Additionally, the review explores emerging strategies for prevention, including the use of wearable devices and genetic testing to identify patients at risk, stem cell therapy and gene therapy for cardiac dysfunction, and precision medicine for personalized treatment plans. Despite some limitations to this review, it provides valuable insights into the current understanding of stroke-related cardiac death and identifies important areas for future research. Ultimately, the implementation of evidence-based prevention strategies and personalized treatment plans has the potential to improve outcomes for patients with stroke and reduce the burden of stroke-related cardiac death in the population.

Nonhomologous end joining during restriction enzyme-mediated DNA integration in Saccharomyces cerevisiae.

The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585-7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5' or 3' overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp718 fragment possessing a 5' overhang integrated into a KpnI (isoschizomer) site possessing a 3' overhang, most likely by filling of the 5' overhang followed by 5' exonuclease digestion to produce a 3' end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.

DNA integration by Ty integrase in yku70 mutant Saccharomyces cerevisiae cells.

In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells.

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo(R) expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo(R) gene flanked by exon 3 of HPRT: The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration approximately 2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.

Poorly repaired mismatches in heteroduplex DNA are hyper-recombinagenic in Saccharomyces cerevisiae.

In yeast meiotic recombination, alleles used as genetic markers fall into two classes as regards their fate when incorporated into heteroduplex DNA. Normal alleles are those that form heteroduplexes that are nearly always recognized and corrected by the mismatch repair system operating in meiosis. High PMS (postmeiotic segregation) alleles form heteroduplexes that are inefficiently mismatch repaired. We report that placing any of several high PMS alleles very close to normal alleles causes hyperrecombination between these markers. We propose that this hyperrecombination is caused by the high PMS allele blocking a mismatch repair tract initiated from the normal allele, thus preventing corepair of the two alleles, which would prevent formation of recombinants. The results of three point crosses involving two PMS alleles and a normal allele suggest that high PMS alleles placed between two alleles that are normally corepaired block that corepair.

Controlled gene expression in mammalian cells via a regulatory cascade involving the tetracycline transactivator and lac repressor.

Regulatory cascades or regulons control pathways at multiple points or multiple genes by one initial signal. In this paper, we describe the construction of an artificial regulatory cascade in CHO cells, which responded to various concentrations of tetracycline (Tc) and/or IPTG. The system consists of the constitutively produced transactivator (TTA) of the Tc operon (tet), which induced the expression of a lacI gene controlled by tet operator (tetO) and upstream CMV promoter (p*CMV) sequences. LacI repressed the activity of a cat gene by binding to lacO sites in its upstream RSV promoter (pRSV) region. However, this repression could be alleviated by exposure to Tc or IPTG, which inhibited the binding activities of TTA and LacI, respectively. Hence, treatment with either Tc or IPTG led to a tenfold increase in CAT activity. After the withdrawal of the inducer, cat expression reverted to basal levels. Regulation by Tc showed a phenotypic lag, and full induction was reached after 192 h, whereas IPTG addition led to full induction within 24 h. When cells were treated with both Tc and IPTG, full induction of cat was reached in 24 h and maintained thereafter while in the presence of Tc alone. This suggests that regulation by Tc is fast and that the phenotypic lag may be due to slow turnover of the LacI repressor. This TTA/lacI regulatory system may serve as an example in which cat expression was used as a reporter. The data indicate that regulatory cascades regulated at multiple points can be constructed with any cloned gene in mammalian cells.