MicroRNA (miRNA) inhibition is a promising therapeutic strategy in several disease indications. MRG-110 is a locked nucleic acid-based antisense oligonucleotide that targets miR-92a-3p and experimentally was shown to have documented therapeutic effects on cardiovascular disease and wound healing. To gain first insights into the activity of anti-miR-92a in humans, we investigated miR-92a-3p expression in several blood compartments and assessed the effect of MRG-110 on target derepression. Healthy adults were randomly assigned (5:2) to receive a single intravenous dose of MRG-110 or placebo in one of seven sequential ascending intravenous dose cohorts ranging from 0.01 to 1.5 mg/kg body weight. MiR-92a-3p whole blood levels were time and dose dependently decreased with halfmaximal inhibition of 0.27 and 0.31 mg/kg at 24 and 72 h after dosing, respectively. In the high-dose groups, >95% inhibition was detected at 24-72 h postinfusion and significant inhibition was observed for 2 weeks. Similar inhibitory effects were detected in isolated CD31 + cells, and miR-92a-3p expression was also inhibited in extracellular vesicles in the high-dose group. Target derepression was measured in whole blood and showed that ITGA5 and CD93 were increased at a dose of 1.5 mg/kg. Single-cell RNA sequencing of peripheral blood cells revealed a cell typespecific derepression of miR-92a targets. Together this study demonstrates that systemic infusion of anti-miR-92a efficiently inhibits miR-92a in the peripheral blood compartment and derepresses miR-92a targets in humans.

Abstract-Treatment of hypertensive patients with β-blockers reduces heart rate and decreases central blood pressure less than other antihypertensive drugs, implying that reducing heart rate without altering brachial blood pressure could increase central blood pressure, explaining the increased cardiovascular risk reported with β-blocker. We describe a randomized, double-blind study to explore whether heart rate reduction with the I f inhibitor ivabradine had an impact on central blood pressure. We included 12 normotensive patients with stable coronary artery disease, heart rate ≥70 bpm (sinus rhythm), and stable background β-blocker therapy. Patients received ivabradine 7.5 mg BID or matched placebo for two 3-week periods with a crossover design and evaluation by aplanation tonometry. Treatment with ivabradine was associated with a significant reduction in resting heart rate after 3 weeks versus no change with placebo (−15. Methods Study Design and PatientsThis randomized, double-blind, crossover study was performed in 3 centers in Paris, Dublin, and Rome. The trial was conducted in accordance with the ethical principles laid out in the Declaration of Helsinki (sixth revision, 2008). Ethics committee approval was obtained in all centers before the start of the study, and all patients provided written informed consent before enrollment. Eligible men and women were outpatients with stable CAD, documented by a history of myocardial infarction, percutaneous coronary intervention, or coronary artery bypass graft at least 3 months before selection; angiographic evidence of ≥50% narrowing of at least 1 major coronary artery; a positive noninvasive stress test, stress echocardiography, or scintigraphic evidence of stress-induced ischemia; or discharge from hospital with a diagnosis of unstable angina within 12 months and at least 3 months before selection. They were aged at least 30 years and were in sinus rhythm with resting heart rate ≥70 bpm on ECG at both selection and inclusion. They had systolic blood pressure/diastolic blood pressure (SBP/DBP) <140/90 mm Hg and normal laboratory values considering their disease. They had been receiving β-blocker for at least 3 months at doses estimated appropriate by the investigator. The original protocol sought to include only patients with type 2 diabetes mellitus but was amended during the study to include nondiabetic patients.Reasons for nonselection or exclusion included a history of central neuropathy, symptomatic orthostatic hypotension, or severe hypotension; treatment with ivabradine; myocardial infarction, revascularization, stroke, or transient ischemic attack in the past 3 months; scheduled for percutaneous coronary intervention or coronary artery bypass graft; patients with a heart transplant, an implanted pacemaker or cardioverter defibrillator, or cardiac resynchronization therapy; valvular disease likely to require surgery within 1 year; permanent atrial fibrillation or flutter, sick sinus syndrome, sinoatrial block, congenital long QT, substance-induced long QT...

Polyelonal antibodies have been raised against two synthetic peptide5 reproducing the 48-64 and 353-369 scqaen~s ofCSK, a protein tyrosine kinase implicated in the down-regulation of sre-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase pr~ent in spleen but they do not cross-react with purified TPK-nB, a spleen protein tyro~ine kina~ sharing with CSK catalytic activity toward src kina~es and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin5epharose affinity chromatography, display opposite specifieities toward synthetic ~ptides reproducing tile sequences around the main phosphoaeceptor residues of pp60 ''~'~, namely Tyr-416 and Tyr-527. The~ data support the view that TPK-IIB and CSK may exert opposite eff~ts on the activity of src-related protein tyrosine kinases.

The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti‐lck antibody, an lck‐like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56‐kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56‐kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription‐PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5′ end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120 to neurons.

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor L L chain (IL-2RL L) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56 lk , one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56 lk phosphorylates in vitro tyrosine residues within the IL-2RL L chain but not those within the IL-2RQ Q chain. p56 lk phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2RL L chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either lk also phosphorylates very efficiently the two tyrosines present in the IL-2RL L chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56 lk with the IL-2RL L chain which was found to depend on a short stretch of the IL-2RL L chain acidic subdomain, and to be independent of the presence of its tyrosine residues.z 1999 Federation of European Biochemical Societies.