Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing.

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of nonuniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs, and nascent polypeptide structure is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

Charting DENR-dependent translation reinitiation uncovers predictive uORF features and links to circadian timekeeping via Clock.

The non-canonical initiation factor DENR promotes translation reinitiation on mRNAs harbouring upstream open reading frames (uORFs). Moreover, DENR depletion shortens circadian period in mouse fibroblasts, suggesting involvement of uORF usage and reinitiation in clock regulation. To identify DENR-regulated translation events transcriptome-wide and, in particular, specific core clock transcripts affected by this mechanism, we have used ribosome profiling in DENR-deficient NIH3T3 cells. We uncovered 240 transcripts with altered translation rate, and used linear regression analysis to extract 5' UTR features predictive of DENR dependence. Among core clock genes, we identified Clock as a DENR target. Using Clock 5' UTR mutants, we mapped the specific uORF through which DENR acts to regulate CLOCK protein biosynthesis. Notably, these experiments revealed an alternative downstream start codon, likely representing the bona fide CLOCK N-terminus. Our findings provide insights into uORF-mediated translational regulation that can regulate the mammalian circadian clock and gene expression at large.

Design of in vitro Transcribed mRNA Vectors for Research and Therapy.

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

Building Bridges between People with Stroke, Families, and Health Professionals: Development of a Blended Care Program for Self-Management.

Evidence-informed interventions for stroke self-management support can influence functional capability and social participation. People with stroke should be offered self-management support after hospital discharge. However, in Portugal, there are no known programs of this nature. This study aimed to develop a person-centered and tailored blended care program for post-stroke self-management, taking into account the existing evidence-informed interventions and the perspectives of Portuguese people with stroke, caregivers, and health professionals. An exploratory sequential mixed methods approach was used, including qualitative methods during stakeholder consultation (stage 1) and co-production (stage 2) and quantitative assessment during prototyping (stage 3). After ethical approval, recruitment occurred in three health units. Results from a literature search led to the adaptation of the Bridges Stroke Self-Management Program. In stage one, 47 participants were interviewed, with two themes emerging: (i) Personalized support and (ii) Building Bridges through small steps. In stage two, the ComVida program was developed, combining in-person and digital approaches, supported by a workbook and a mobile app. In stage three, 56 participants evaluated prototypes, demonstrating a strong level of quality. Understandability and actionability of the developed tools obtained high scores (91-100%). The app also showed good usability (A-grade) and high levels of recommendation (5 stars).

Percepção do farmacêutico analista clínico: da formação à atuação profissional / Perception of pharmaceutical clinical analyst: from academic education to professional performance

A atuação do farmacêutico em análises clínicas está diretamente relacionada com o estudo e o diagnóstico da saúde do paciente. O presente artigo objetivou compreender a percepção de farmacêuticos analistas clínicos em relação à sua formação acadêmica. A pesquisa contou com a participação de farmacêuticos que atuam como analistas clínicos em um laboratório. Os dados coletados foram analisados pela técnica de análise de conteúdo e divididos em duas categorias: formação acadêmica e formação em análises clínicas. O resultado mostra que as aulas práticas durante a graduação são de suma importância, pois propiciam que o futuro farmacêutico analista clínico desenvolva habilidades e atitudes atreladas ao conhecimento. Constatou-se também que os conhecimentos teóricos propiciam o embasamento para a prática e que a formação do farmacêutico analista clínico deve priorizar o contexto em que estará inserido de forma proativa na equipes multiprofissionais e na sociedade.

The work of the medical laboratory scientist is directly related to the study and diagnosis of the patient's health. This study aimed to understand the opinion of medical laboratory scientists regarding their academic training. The research had the participation of pharmacists who work as medical laboratory scientists. The collected data were analyzed using the content analysis technique and divided into two categories: academic training and clinical analysis training. The result shows that practical classes during the undergraduate course are of paramount importance, as they allow the future medical laboratory scientist to develop skills and attitudes linked to knowledge. It was also found that theoretical knowledge provides the basis for the practice and that the training of the medical laboratory scientist shall prioritize the context where he/she will be proactively inserted in the multiprofessional teams and in society.

El desempeño del farmacéutico en el análisis clínico está directamente relacionado con el estudio y el diagnóstico de la salud del paciente. Este artículo tuvo como objetivo comprender la percepción de los analistas clínicos farmacéuticos en relación con sus antecedentes académicos. A la investigación asistieron farmacéuticos que actúan como analistas clínicos en un laboratorio. Los datos recopilados se analizaron utilizando la técnica de análisis de contenido y se dividieron en dos categorías: capacitación académica y capacitación en análisis clínico. El resultado muestra que las clases prácticas durante la graduación son de suma importancia, ya que permiten al futuro analista clínico farmacéutico desarrollar habilidades y actitudes vinculadas al conocimiento. También se descubrió que el conocimiento teórico proporciona la base para la práctica y que la formación del analista clínico farmacéutico debe priorizar el contexto en el que se insertará proactivamente en los equipos multiprofesionales y en la sociedad.

MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale.

A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.DOI: http://dx.doi.org/10.7554/eLife.02510.001.

O uso da toxina botulínica na correção do sorriso gengival- revisão de literatura / Use of the botulinum toxin in correction of the gingival smile - Literature review

A toxina botulínica inicialmente era utilizada apenas para tratamentos terapêuticos, depois de alguns estudos sua aplicação passou a ser também utilizada para tratamentos estéticos. Esta substância é produzida pela bactéria Clostridium botulinum, responsável pelo botulismo, doença que provoca intoxicação por alimentos mal conservados. Utilizada na medicina para tratar inicialmente patologias, hoje seu uso passa a ser também estético na odontologia. Alguns pacientes se queixam do sorriso gengival causado por uma exposição excessiva da gengiva e buscam tratamentos mais rápidos e com menor morbidade, neste caso a toxina botulínica é muito eficaz para a correção do sorriso gengival diagnosticado por uma hiperfunção muscular. Quando causado por excesso vertical maxilar, extrusão ou erupção passiva alterada dos dentes ântero-superiores, o sorriso gengival pode ser tratado com a cirurgia ortognática, tratamento ortodôntico, miectomia, reposicionamento labial e/ou gengivoplastia. O uso desta substância na odontologia, pode ser mais uma opção para amenizar ou corrigir o sorriso gengival por hiperfunção muscular ou associar a outros tipos de tratamentos. Desta forma, o objetivo deste trabalho é fazer uma revisão literária sobre o uso da toxina botulínica na correção do sorriso gengival e descrever seu protocolo de uso (AU)

Botulinum toxin initially was used only for therapeutic treatments, after some studies its application was also used for aesthetic treatments. This substance was produced by the bacterium Clostridium botulinum, responsible for botulism, a disease that causes intoxication by poorly preserved foods. Used in medicine to treat initially pathologies, today its use is now also aesthetic in dentistry. Some patients complain of the gingival smile caused by excessive exposure of the gums and seek faster treatments with lower morbidity; in this case the botulinum toxin is very effective for the correction of the gingival smile diagnosed by a muscular hyperfunction. When caused by excessive vertical jaw, extrusion or passive eruption of the upper anterior teeth, the gingival smile may be treated with orthognathic surgery, orthodontic treatment, myectomy, lip repositioning and / or gingivoplasty. The use of this substance in dentistry may be another option to soften or correct the gingival smile due to muscular hyperfunction or to associate with other types of treatments. In this way, the objective of this work is to make a literary review on the use of botulinum toxin in the correction of the gingival smile and to describe its protocol of use. (AU)