Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/-) mice.

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Programa de seguimiento al alta de la unidad de cuidados intensivos / Follow-up program after intensive care unit discharge

Introducción: El seguimiento de pacientes al alta de la unidad de cuidados intensivos (UCI) permite el reconocimiento precoz de las complicaciones asociadas al síndrome post terapia intensiva (SPTI). El objetivo de este estudio es la estandarización de medidas básicas de resultado incluidas en un programa de seguimiento de pacientes en riesgo de presentar SPTI. Métodos: El comité de Seguimiento y Rehabilitación luego de la Enfermedad Crítica de la Sociedad Argentina de Terapia Intensiva (SATI) convocó a diferentes comités para la redacción del presente documento. Se realizó una búsqueda bibliográfica, junto con reuniones y foros de discusión. Las recomendaciones incluidas en el programa de seguimiento según el sistema GRADE son: frecuencia de seguimiento, profesionales encargados del programa, dominios básicos a evaluar, herramientas recomendadas para su evaluación validadas al español y duración del programa. Conclusión: Las medidas recomendadas para el seguimiento de pacientes permiten establecer los lineamientos básicos para la identificación y tratamiento precoz de las complicaciones asociadas al SPTI

Introduction: Patient follow-up after intensive care unit (ICU) discharge allows the early recognition of complications associated to post-intensive care syndrome (PICS). The aim of this project is to standardize outcome variables in a follow-up program for patients at risk of suffering PICS. Methods: The Rehabilitation and Patient Follow-up Committee of the Argentine Society of Intensive Care Medicine (Sociedad Argentina de Terapia Intensiva, SATI) requested the collaboration of different committees to design the present document. A thorough search of the literature on the issue, together with pre-scheduled meetings and web-based discussion encounters were carried out. After comprehensive evaluation, the recommendations according to the GRADE system included in the follow-up program were: frequency of controlled visits, appointed healthcare professionals, basic domains of assessment and recommended tools of evaluation, validated in Spanish, and entire duration of the program. Conclusion: The measures herein suggested for patient follow-up after ICU discharge will facilitate a basic approach to diagnosis and management of the long-term complications associated to PICS

Follow-up program after intensive care unit discharge. / Programa de seguimiento al alta de la unidad de cuidados intensivos.

INTRODUCTION: Patient follow-up after intensive care unit (ICU) discharge allows the early recognition of complications associated to post-intensive care syndrome (PICS). The aim of this project is to standardize outcome variables in a follow-up program for patients at risk of suffering PICS. METHODS: The Rehabilitation and Patient Follow-up Committee of the Argentine Society of Intensive Care Medicine (Sociedad Argentina de Terapia Intensiva, SATI) requested the collaboration of different committees to design the present document. A thorough search of the literature on the issue, together with pre-scheduled meetings and web-based discussion encounters were carried out. After comprehensive evaluation, the recommendations according to the GRADE system included in the follow-up program were: frequency of controlled visits, appointed healthcare professionals, basic domains of assessment and recommended tools of evaluation, validated in Spanish, and entire duration of the program. CONCLUSION: The measures herein suggested for patient follow-up after ICU discharge will facilitate a basic approach to diagnosis and management of the long-term complications associated to PICS.

miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure.

MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.

Cytokine-Like 1 Regulates Cardiac Fibrosis via Modulation of TGF-ß Signaling.

Cytokine-like 1 (Cytl1) is a secreted protein that is involved in diverse biological processes. A comparative modeling study indicated that Cytl1 is structurally and functionally similar to monocyte chemoattractant protein 1 (MCP-1). As MCP-1 plays an important role in cardiac fibrosis (CF) and heart failure (HF), we investigated the role of Cytl1 in a mouse model of CF and HF. Cytl1 was upregulated in the failing mouse heart. Pressure overload-induced CF was significantly attenuated in cytl1 knock-out (KO) mice compared to that from wild-type (WT) mice. By contrast, adeno-associated virus (AAV)-mediated overexpression of cytl1 alone led to the development of CF in vivo. The endothelial-mesenchymal transition (EndMT) and the transdifferentiation of fibroblasts (FBs) to myofibroblasts (MFBs) have been suggested to contribute considerably to CF. Adenovirus-mediated overexpression of cytl1 was sufficient to induce these two critical CF-related processes in vitro, which were completely abrogated by co-treatment with SB-431542, an antagonist of TGF-ß receptor 1. Cytl1 induced the expression of TGF-ß2 both in vivo and in vitro. Antagonizing the receptor for MCP-1, C-C chemokine receptor type 2 (CCR2), with CAS 445479-97-0 did not block the pro-fibrotic activity of Cytl1 in vitro. Collectively, our data suggest that Cytl1 plays an essential role in CF likely through activating the TGF-ß-SMAD signaling pathway. Although the receptor for Cyt1l remains to be identified, Cytl1 provides a novel platform for the development of anti-CF therapies.

Prevalence, molecular characterization, and phenotypic confirmation of extended-spectrum beta-lactamases in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca at the Radboud University Nijmegen Medical Centre in The Netherlands.

The prevalence and molecular types of extended-spectrum beta-lactamases (ESBLs) were determined during a 1-year period in unselected clinical nonduplicate isolates of Escherichia coli (n = 1,738), Klebsiella pneumoniae (n = 436), and Klebsiella oxytoca (n = 208), cultured at the University Medical Centre Nijmegen, The Netherlands. Isolates identified as ESBL producer by the Phoenix automated system were collected prospectively and subjected to molecular analysis for the most common ESBLs TEM, SHV, and CTX-M, as well as OXA and GES. Both the Etest ESBL and double-disk synergy test were performed as confirmatory tests. The estimated prevalence of ESBLs was 2.1% in E. coli, 5.2% in K. pneumoniae, and 2.4% in K. oxytoca. TEM-12 and -26, SHV-5 and -12, and CTX-M groups 1 and 9 were the most frequent ESBLs found. Isolates identified as ESBLs by the Phoenix were confirmed by polymerase chain reaction (PCR) in only 42%. In ESBL PCR-positive E. coli and K. pneumoniae, both confirmatory tests were positive in 95% of the isolates. In 28% of the Etest and 13% of the double-disk synergy test-positive isolates, PCR could not detect any ESBL gene. In these cases, other resistance mechanisms may play a role. Confirmatory tests were unreliable for K. oxytoca. A previously described mutation in the K1 enzyme was detected in one ceftazidime-resistant K. oxytoca. The prevalence of ESBLs in The Netherlands is increasing. The predominant molecular types of ESBLs detected were comparable to other studies. Phoenix ESBL results need to be confirmed as advocated by ESBL detection guidelines.

An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+.

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.