Activation of recombinases at specific DNA loci by zinc-finger domain insertions.

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.

Adding value to the chia (Salvia hispanica L.) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain.

Chia expeller is a by-product of the extrusion process of chia seeds generated during oil production. Typically, this material is non-utilized or used for non-valuable applications. In the present work, the chia expeller was hydrolysed with Papain and the antioxidant properties of the resultant peptides were evaluated. Papain treatment of the chia seed expeller demonstrated an enrichment of low molecular weight peptides (molecular weight <15 kDa) as determined by SDS-PAGE and MALDI-TOF/MS analyses. Such peptides showed a potent radical scavenging effect in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals in comparison with those non-hydrolysed samples. Taken together our results demonstrate the generation of functional peptides from the chia expeller by enzymatic hydrolysis with Papain. This value-added hydrolysate can be potentially included as a supplement in functional food and nutraceutical products.

Biochemical characterization of the YBPCI miniprotein, the first carboxypeptidase inhibitor isolated from Yellow Bell Pepper (Capsicum annuum L). A novel contribution to the knowledge of miniproteins stability.

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 µg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance.

Lesson From the New York City Out-of-Hospital Uncontrolled Donation After Circulatory Determination of Death Program.

STUDY OBJECTIVE: In 2006, the Institute of Medicine emphasized substantial potential to expand organ donation opportunities through uncontrolled donation after circulatory determination of death (uDCDD). We pilot an out-of-hospital uDCDD kidney program for New York City in partnership with communities that it was intended to benefit. We evaluate protocol process and outcomes while identifying barriers to success and means for improvement. METHODS: We conducted a prospective, participatory action research study in Manhattan from December 2010 to May 2011. Daily from 4 to 12 pm, our organ preservation unit monitored emergency medical services (EMS) frequencies for cardiac arrests occurring in private locations. After EMS providers independently ordered termination of resuscitation, organ preservation unit staff determined clinical eligibility and donor status. Authorized parties, persons authorized to make organ donation decisions, were approached about in vivo preservation. The study population included organ preservation unit staff, authorized parties, passersby, and other New York City agency personnel. Organ preservation unit staff independently documented shift activities with daily operations notes and teleconference summaries that we analyzed with mixed qualitative and quantitative methods. RESULTS: The organ preservation unit entered 9 private locations; all the deceased lacked previous registration, although 4 met clinical screening eligibility. No kidneys were recovered. We collected 837 notes from 35 organ preservation unit staff. Despite frequently recounting protocol breaches, most responses from passersby including New York City agencies were favorable. No authorized parties were offended by preservation requests, yielding a Bayesian posterior median 98% (95% credible interval 76% to 100%). CONCLUSION: In summary, the New York City out-of-hospital uDCDD program was not feasible. There were frequent protocol breaches and confusion in determining clinical eligibility. In the small sample of authorized persons we encountered during the immediate grieving period, negative reactions were infrequent.

A peer-to-peer campaign to promote organ donation among racially diverse college students in New York City.

PURPOSE: Due to the documented disparities in organ transplantation, individuals from racially diverse backgrounds are encouraged to register as donors and communicate their donation intentions to family. The present study reports an intervention aimed at addressing students' fears and misconceptions related to organ transplantation on college campuses with racially diverse student enrollments. The intervention uses peer-to-peer campaigns at colleges in New York City in an attempt to increase declarations of intent to donate and to educate students about transplantation. PROCEDURES: Six campuses in the New York City area participated in the intervention. Students participating in the college campaign intervention were educated about donation through active learning methods and implemented campus-wide campaigns to recruit fellow students as donors. RESULTS: Student campaigners reported increases in topic salience and self-efficacy from precampaign to postcampaign activities. In addition, rates of donor registration and family communication improved over time. Across 6 campuses, campaigners were able to recruit 1019 students as registered donors. CONCLUSIONS: Participation in the campaign course increases students' vested interest in donation and has proven effective at increasing donor registration among racially diverse groups.

Detección de plesiomonas shigelloides mediante la pcr en tilapias silvestres (oreochromis mossambicus) y cultivadas (tetrahíbrido o. Mossambicus × o. Urolepis hornorum × o. Niloticus × o. Aureus) en venezuela / Detection of plesiomonas shigelloides by pcr in feral (oreochromis mossambicus) and cultured tilapias (tetrahybrid of o. Mossambicus × o. Urolepis hornorum × o. Niloticus × o. Aureus) in venezuela

Plesiomonas shigelloides es un bacilo Gram negativo, anaerobio facultativo, móvil, que pertenece a la familia Enterobactereaceae. No forma parte de la flora normal del humano, mientras que los peces dulceacuícolas como las tilapias se tienen como su reservorio primario. Considerando: a) La implicación cada vez más frecuente de este microorganismo como agente causal de enfermedades intra y extra intestinales, b) El incremento de la comercialización de tilapias silvestres y de cultivo a nivel nacional y c) La necesidad de métodos de detección e identificación rápidos y efectivos de patógenos, se aplicó el método biomolecular de la Reacción en Cadena de la Polimerasa (PCR). Se procesaron mediante la PCR, cepas de P. shigelloides y homogeneizados de pools de bazo, riñón y cerebro, y de intestino de tilapias, utilizándose oligonucleótidos iniciadores específicos para el gen 23S ARNr de P. shigelloides. Se obtuvo el reconocimiento del 75% de las 20 cepas identificadas por pruebas fenotípicas convencionales, detectándose además en 27% de los homogeneizados frescos y en 18% de los congelados. Estos resultados confirman la especificidad del método para la identificación de diversas bacterias potencialmente patógenas, su utilidad en el mejoramiento del sistema de control de calidad de granjas dulceacuícolas y en el estudio de casos clínicos, aumentando la efectividad del sistema de salud relacionado con estas áreas.

Plesiomonas shigelloides is a Gram negative rod, facultative anaerobic, motile, that belongs to the Enterobacteriaceae. It’s not part of the normal human flora, while fresh water fish like tilapias are thought to be its primary reservoir. Considering: a) The more frequent implication of this microorganism as the causal agent of intra and extraintestinal infections, b) The wide spread of national commercialization of feral and cultured tilapias and c) The need for rapid and effective detection and identification methods, the biomolecular technique of Polymerase Chain Reaction (PCR) was applied. Strains of P. shigelloides and pools of homogenates from spleen, kidney and brain and the intestine of tilapias were processed by PCR, using specific primers for the gene 23S rRNA of P. shigelloides. The 20 strains identified by conventional phenotypic traits were identified in 75%, additionally it was detected in 27% of the fresh homogenates and in 18% of the frozen ones. These results confirm the specificity of the technique for the identification of a diverse range of potential pathogenic bacteria, its usefulness in the improvement of quality control in freshwater farms and in the study of clinical cases, improving the effectiveness of health systems related with these areas.

La participación social en salud de los trabajadores ambulantes / The social participation to health of the ambulant workers

Busca transmitir un conjunto de experiencias de participación social para la gestión en salud llevadas a cabo por trabajadores ambulantes, de manera especial aquellas que desarrollan las promotoras de salud, en particular las referidas a su acción frente a la epidemia del cólera en el Cono Norte; reflexiona en torno a la propia organización, sus limitaciones y perspectivas