Polysaccharide-Polyplex Nanofilm Coatings Enhance Nanoneedle-Based Gene Delivery and Transfection Efficiency.

Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.

Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.

The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.

Production and Purification of Supercoiled Minicircles by a Combination of In Vitro Endonuclease Nicking and Hydrophobic Interaction Chromatography.

A wider application of minicircle (MC) vectors in gene therapy research depends critically on the ability to purify supercoiled (sc) MC from related miniplasmid (MP) and parental plasmid (PP) impurities. This protocol describes a purification strategy that combines the in vitro enzymatic relaxation of sc MP and PP impurities by a nicking endonuclease, and topoisomer separation and RNA clearance by hydrophobic interaction chromatography. The time required to follow the full protocol, from production to isolation of sc MC, is approximately 50 h. The process delivers sc MCs that are virtually free from MP, PP, RNA, and protein impurities.

Procedimientos metodológicos para la implementación de la estrategia curricular Salud Pública y Educación Ambiental / Methodological procedures for the implementation of the curricular strategy Public Health and Environmental Education

Fundamento: las insuficiencias en la implementación de la estrategia curricular Salud Pública y Educación Ambiental en la formación del estudiante de Medicina, limitan la formación salubrista del médico general. Objetivo: elaborar procedimientos metodológicos traducidos en acciones concretas para perfeccionar la implementación de la estrategia curricular Salud Pública y Educación Ambiental. Método: se desarrolló una investigación de desarrollo esencialmente cualitativa desde noviembre 2016 a septiembre 2017 en la Filial de Ciencias Médicas de Puerto Padre, de la Universidad de Ciencias Médicas de Las Tunas. Se utilizaron métodos teóricos: análisis-síntesis e inducción-deducción; y empíricos: encuesta en forma de cuestionario a docentes y estudiantes. Resultados: se constataron carencias en cuanto al conocimiento de la metodología para aplicar la estrategia curricular Salud Pública y Educación Ambiental en los docentes, las cuales conspiran contra su efectiva implementación en las diferentes disciplinas, asignaturas y estancias desde primero a quinto años de la carrera de Medicina; esto limita el desarrollo de habilidades en los estudiantes para su futuro desempeño como médicos generales, por lo que se diseñaron procedimientos metodológicos. Conclusiones: fueron valorados por especialistas como pertinentes, factibles de aplicar y de utilidad para la ejecución de la mencionada estrategia curricular, por lo que constituye una herramienta adecuada en la formación salubrista del médico general.

Background: the insufficiencies in the implementation of the curricular strategy Public Health and Environmental Education in the training of the medical student, limit the training of the general practitioner. Objective: to develop methodological procedures that lead to concrete actions to improve the implementation of the curricular strategy Public Health and Environmental Education. Method: an essentially qualitative development research work was developed from November 2016 to September 2017 in the Medical Sciences site of Puerto Padre, of Las Tunas Medical Sciences University. Theoretical methods were used: analysis-synthesis, induction-deduction and the historical-logical; and empirical ones: survey in the form of a questionnaire for teachers and students. Results: deficiencies were found in the knowledge of the methodology to apply the curricular strategy Public Health and Environmental Education in the teachers, which conspire against its effective implementation in the different disciplines, subjects and rotations from the first to fifth years of the career of Medicine; this limits the development of skills in the students for their future performance as general practitioners, that's why methodological procedures were designed. Conclusions: they were valued by specialists as relevant, feasible to apply and useful for the implementation of the aforementioned curricular strategy, for that reason constitutes an adequate tool in the general practitioner's health education.

Plantão psicológico: ampliando possibilidades de escuta / Psychological care service: widening listening possibilities

O Plantão Psicológico, enquanto uma modalidade clínica contemporânea que se caracteriza por realizar atendimentos psicoterapêuticos de caráter emergencial destinados à comunidade que a ele recorre espontaneamente, sem a necessidade de agendamento prévio, foi reimplantado em meados de 2015 na Clínica Escola da UFC. Servindo como espaço de acolhimento e de informação e auxiliando as pessoas a terem uma maior autonomia emocional, o Plantão Psicológico vem sendo realizado por estagiários e extensionistas, desenvolvendo e consolidando a partir da sedimentação desta modalidade clínica, o caráter necessariamente transdisciplinar da Clínica Escola. O projeto de extensão do Plantão Psicológico prioriza a qualificação da formação dos discentes do curso de Psicologia com a vivência de diferentes experiências frente às demandas variadas; o fortalecimento de parcerias com instituições de saúde do Estado e, sobretudo, a otimização da fila de espera da Clínica Escola, promovendo atendimento imediato e de qualidade. Acredita-se que o Plantão consolida o ensino, a pesquisa e a extensão no âmbito da universidade, estabelecendo diálogo e intervenção efetiva junto à comunidade em geral. Trata-se do processo de ampliação das possibilidades de escuta clínica que, gradativamente, vem tornando o Plantão Psicológico da Clínica Escola da UFC uma referência no Estado do Ceará.

The Psychological Care Service, while a contemporary clinical modality that is characterized by performing psychotherapeutic calls of an emergency nature for the community that uses it spontaneously, without the need to schedule in advance, was redeployed in mid 2015 at the Clinical School of UFC. Serving as host space and information and helping people to have greater emotional autonomy, psychological Plantão being done by trainees and extension, developing and consolidating from the consolidation of this clinical modality, necessarily transdisciplinary nature of the Clinical School. The Psychological Care Service extension project prioritizes qualification training of students of the Psychology course with the experience of different experiences with the different demands; strengthening partnerships with health institutions of the state and, above all, the optimization of the queue of Clinical School, providing immediate service and quality. It is believed that the Psychological Care Service consolidates teaching, research and extension within the university, establishing dialogue and effective intervention by the community at large. This is the process of enlarging clinical listening possibilities that gradually is making the Psychological Clinic Duty UFC School a reference in the State of Ceará.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.

Development of a nicking endonuclease-assisted method for the purification of minicircles.

Minicircle (MC) DNA vectors are able to generate a high-level transgene expression in vivo, which is superior to the one afforded by conventional plasmids. MC vectors are produced by replicating a parental plasmid (PP) and promoting its recombination in Escherichia coli. This generates a MC with the expression cassette, and a miniplasmid (MP) with the replication segment. Unfortunately, wider use of MC vectors is hampered by difficulties in isolating the target MCs from their MP counterpart. In this proof-of-concept study, a reproducible process is described to improve the purification of supercoiled (sc) MCs that combines an in vitro enzymatic relaxation of sc MP impurities with topoisomer separation and RNA clearance by hydrophobic interaction chromatography (HIC) step. At the early stage of vector design, a site for the nicking endonuclease Nb.BbvCI was strategically placed in the MP part of the PP backbone. A process was then established that involves E. coli culture and recombination of PPs into target MC, cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. Next, an in vitro digestion step was carried out with Nb.BbvCI to nick of one of the strands of the MPs and of non-recombined PPs by Nb.BbvCI. As a result, sc MPs and non-recombined PPs were converted into the corresponding open circular (oc) forms whereas sc MCs remain unaffected. Finally, sc MC was isolated from oc DNA molecules (oc MPs, oc MC) and RNA by performing HIC with a phenyl-Sepharose column using a series of elution steps with decreasing ammonium sulfate concentrations. On the basis of agarose gel electrophoresis analysis, the sc MC-containing fractions were determined to be virtually free from nucleic acid impurities.

Absence of tmRNA Has a Protective Effect against Fluoroquinolones in Streptococcus pneumoniae.

The transfer messenger RNA (tmRNA), encoded by the ssrA gene, is a small non-coding RNA involved in trans-translation that contributes to the recycling of ribosomes stalled on aberrant mRNAs. In most bacteria, its inactivation has been related to a decreased ability to respond to and recover from a variety of stress conditions. In this report, we investigated the role of tmRNA in stress adaptation in the human pathogen Streptococcus pneumoniae. We constructed a tmRNA deletion mutant and analyzed its response to several lethal stresses. The ΔssrA strain grew slower than the wild type, indicating that, although not essential, tmRNA is important for normal pneumococcal growth. Moreover, deletion of tmRNA increased susceptibility to UV irradiation, to exogenous hydrogen peroxide and to antibiotics that inhibit protein synthesis and transcription. However, the ΔssrA strain was more resistant to fluoroquinolones, showing twofold higher MIC values and up to 1000-fold higher survival rates than the wild type. Deletion of SmpB, the other partner in trans-translation, also reduced survival to levofloxacin in a similar extent. Accumulation of intracellular reactive oxygen species associated to moxifloxacin and levofloxacin treatment was also highly reduced (∼100-fold). Nevertheless, the ΔssrA strain showed higher intracellular accumulation of ethidium bromide and levofloxacin than the wild type, suggesting that tmRNA deficiency protects pneumococcal cells from fluoroquinolone-mediated killing. In fact, analysis of chromosome integrity revealed that deletion of tmRNA prevented the fragmentation of the chromosome associated to levofloxacin treatment. Moreover, such protective effect appears to relay mainly on inhibition of protein synthesis, since a similar effect was observed with antibiotics that inhibit that process. The emergence and spread of drug-resistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.