I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy

Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) has been noted to produce ischemic preconditioning (IPC)-mediated cardioprotection. Caveolin is a negative regulator of NO, which inhibits endothelial nitric oxide synthase (eNOS) by making caveolin-eNOS complex. The expression of caveolin is increased during diabetes mellitus (DM). The present study was designed to investigate the involvement of caveolin in attenuation of the cardioprotective effect of IPC during DM in rat.</p> <p>Methods</p> <p>Experimental DM was induced by single dose of streptozotocin (50 mg/Kg, <it>i.p</it>,) and animals were used for experiments four weeks later. Isolated heart was mounted on Langendorff's apparatus, and was subjected to 30 min of global ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Kreb's-Henseleit solution (K-H). Extent of injury was measured in terms of infarct size by triphenyltetrazolium chloride (TTC) staining, and release of lactate dehydrogenase (LDH) and creatin kinase-MB (CK-MB) in coronary effluent. The cardiac release of NO was noted by measuring the level of nitrite in coronary effluent.</p> <p>Results</p> <p>IPC- induced cardioprotection and release of NO was significantly decreased in diabetic rat heart. Pre-treatment of diabetic rat with daidzein (DDZ) a caveolin inhibitor (0.2 mg/Kg/s.c), for one week, significantly increased the release of NO and restored the attenuated cardioprotective effect of IPC. Also perfusion of sodium nitrite (10 μM/L), a precursor of NO, significantly restored the lost effect of IPC, similar to daidzein in diabetic rat. Administration of 5-hydroxy deaconate (5-HD), a mito K_ATPchannel blocker, significantly abolished the observed IPC-induced cardioprotection in normal rat or daidzein and sodium nitrite perfused diabetic rat heart alone or in combination.</p> <p>Conclusions</p> <p>Thus, it is suggested that attenuation of the cardioprotection in diabetic heart may be due to decrease the IPC mediated release of NO in the diabetic myocardium, which may be due to up -regulation of caveolin and subsequently decreased activity of eNOS.</p

Revascularization for coronary artery disease in diabetes mellitus: Angioplasty, stents and coronary artery bypass grafting

Author Manuscript: 2011 April 14Patients with diabetes mellitus (DM) are prone to a diffuse and rapidly progressive form of atherosclerosis, which increases their likelihood of requiring revascularization. However, the unique pathophysiology of atherosclerosis in patients with DM modifies the response to arterial injury, with profound clinical consequences for patients undergoing percutaneous coronary intervention (PCI). Multiple studies have shown that DM is a strong risk factor for restenosis following successful balloon angioplasty or coronary stenting, with greater need for repeat revascularization and inferior clinical outcomes. Early data suggest that drug eluting stents reduce restenosis rates and the need for repeat revascularization irrespective of the diabetic state and with no significant reduction in hard clinical endpoints such as myocardial infarction and mortality. For many patients with 1- or 2-vessel coronary artery disease, there is little prognostic benefit from any intervention over optimal medical therapy. PCI with drug-eluting or bare metal stents is appropriate for patients who remain symptomatic with medical therapy. However, selection of the optimal myocardial revascularization strategy for patients with DM and multivessel coronary artery disease is crucial. Randomized trials comparing multivessel PCI with balloon angioplasty or bare metal stents to coronary artery bypass grafting (CABG) consistently demonstrated the superiority of CABG in patients with treated DM. In the setting of diabetes CABG had greater survival, fewer recurrent infarctions or need for re-intervention. Limited data suggests that CABG is superior to multivessel PCI even when drug-eluting stents are used. Several ongoing randomized trials are evaluating the long-term comparative efficacy of PCI with drug-eluting stents and CABG in patients with DM. Only further study will continue to unravel the mechanisms at play and optimal therapy in the face of the profoundly virulent atherosclerotic potential that accompanies diabetes mellitus.National Institutes of Health (U.S.) (GM 49039

Trends in Pretreatment HIV-1 Drug Resistance in Antiretroviral Therapy-naive Adults in South Africa, 2000–2016: A Pooled Sequence Analysis

Background: South Africa has the largest public antiretroviral therapy (ART) programme in the world. We assessed temporal trends in pretreatment HIV-1 drug resistance (PDR) in ART-naïve adults from South Africa. Methods: We included datasets from studies conducted between 2000 and 2016, with HIV-1 pol sequences from more than ten ART-naïve adults. We analysed sequences for the presence of 101 drug resistance mutations. We pooled sequences by sampling year and performed a sequence-level analysis using a generalized linear mixed model, including the dataset as a random effect. Findings: We identified 38 datasets, and retrieved 6880 HIV-1 pol sequences for analysis. The pooled annual prevalence of PDR remained below 5% until 2009, then increased to a peak of 11·9% (95% confidence interval (CI) 9·2-15·0) in 2015. The pooled annual prevalence of non-nucleoside reverse-transcriptase inhibitor (NNRTI) PDR remained below 5% until 2011, then increased to 10.0% (95% CI 8.4–11.8) by 2014. Between 2000 and 2016, there was a 1.18-fold (95% CI 1.13–1.23) annual increase in NNRTI PDR (p < 0.001), and a 1.10-fold (95% CI 1.05–1.16) annual increase in nucleoside reverse-transcriptase inhibitor PDR (p = 0.001). Interpretation: Increasing PDR in South Africa presents a threat to the efforts to end the HIV/AIDS epidemic. These findings support the recent decision to modify the standard first-line ART regimen, but also highlights the need for broader public health action to prevent the further emergence and transmission of drug-resistant HIV. Source of Funding: This research project was funded by the South African Medical Research Council (MRC) with funds from National Treasury under its Economic Competitiveness and Support Package. Disclaimer: The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CDC

Hemodynamic effects of a new antiarrhythmic agent, flecainide (R-818), in coronary heart disease.

The hemodynamic effects of flecainide acetate, a new class I antiarrhythmic agent, were studied in 10 patients with coronary heart disease. The drug was injected intravenously at a dose of 2 mg/kg over 30 minutes. The mean drug plasma level achieved was 394 ng/ml (range 329 to 470). The heart rate did not change, but a significant increase (p less than 0.001) in P-R (+17%), QRS (+15%), and Q-T (+7%) duration occurred after drug administration. Negative inotropic effects also were observed and consisted of an increase (p less than 0.01) in pulmonary wedge pressure (+27%) and a decrease (p less than 0.01) in stroke index (-10%), left ventricular stroke work index (-12%), and left ventricular ejection rate (-11%). No significant change in mean aortic pressure or systemic and pulmonary vascular resistance occurred. Left ventriculography performed after drug infusion revealed a significant increase (p less than 0.01) in systolic volume (+9%) and a decrease in ejection fraction (-9%) and mean velocity of circumferential fiber shortening (Vcf) (-13%). A progressive and significant decrease of dP/dt was observed during drug infusion, but 15 minutes after the injection, dP/dt had returned to near basal values. Thus, flecainide acetate has slight, but significant negative inotropic effects, particularly conspicuous during drug infusion. The drug should be administered with caution in patients with poorly compensated heart

Haemodynamic effects of intravenous diltiazem at rest and exercise in patients with coronary artery disease.

The acute effects of intravenous diltiazem on exercise performance were studied in 10 patients with coronary artery disease. Haemodynamic measurements were made at rest and during exercise before and after 0.5 mg kg-1 of diltiazem. Diltiazem prolonged the duration of exercise (+2.85 min, P less than 0.001) and delayed the onset of ischaemic ST depression or angina in all patients. The highest tolerated heart rate and pressure rate product were increased in all but one patient after diltiazem. At rest diltiazem decreased mean arterial pressure (-10.8%, P less than 0.005), systemic vascular resistance (SVR) (-11.8%, P less than 0.05) and left ventricular stroke work index (SWI) (-14.1%, P less than 0.005). During exercise under diltiazem therapy, at the level achieved before the drug, the pulmonary capillary wedge pressure (-30%, P less than 0.005) and the SVR (-13.6%, P less than 0.02) were lowered, the SWI (+13%, P less than 0.01) was increased; at the end of exercise only the SVR (-14%, P less than 0.05) was reduced. Two patients experienced angina on lying down and one had orthostatic hypotension after exercise with diltiazem. This study indicates that intravenous diltiazem is a potentially useful agent for the treatment of angina by reducing myocardial oxygen demand at rest and by improving left ventricular performances on exercise

Comparative haemodynamic effects of intravenous flecainide in patients with and without heart failure and with and without beta-blocker therapy.

The haemodynamic effects of flecainide were compared in three different subsets of patients with documented coronary disease. Ten patients (A) had no heart failure, 5 patients were on beta blockers (B) and 5 patients had overt heart failure (C). Flecainide was associated with negative inotropic effects that were relatively more pronounced in patients with left ventricular dysfunction: pulmonary wedge pressure increased by 27% in A, by 31% in B and by 42% in C; left ventricular stroke volume and stroke work decreased respectively by 10 and 12% in A, 21 and 19% in B, 26 and 28% in C. Ejection fraction decreased by 9% in A, 13% in B and 20% in C, in relation with an increase in end systolic volume (+9% in A, +10% in B and +5% in C). Absolute changes, however, were not significantly different from one group to another except for the increase of systemic vascular resistance which was more pronounced in C as compared with the other groups. The myocardial depression was also confirmed by the fall in dP/dt that was maximal at the end of injection; dP/dt remained depressed 15 min later despite some improvement. Flecainide thus exerts negative inotropic effects that are maximal at the end of infusion and may be of importance in patients with established left ventricular dysfunction