An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

Arquivo de Memória Oral das Profissões da Comunicação (AMOPC)

O AMOPC designa um repositório de memórias orais dos profissionais das várias áreas da comunicação. O objetivo é a criação de um arquivo audiovisual em formato eletrónico, para recolha, recuperação, catalogação, preservação, disponibilização e aproveitamento de registos de memória histórica sobre as profissões da comunicação, aberto à sociedade civil e à comunidade académica mais alargada. O AMOPC poderá ser acedido e pesquisável através da internet, permitindo a rentabilização de materiais que se constituirão como fonte permanente de informação pedagógica e científica. Os media constituem um dos elementos mais determinantes das sociedades contemporâneas. À medida que à escala mundial se vai constituindo um híper sector, assistindo-se a fenómenos de convergência e de homogeneização cultural, os estudos na área da comunicação assumem um papel relevante para a compreensão das reconfigurações da esfera pública. Face à mudança nas atividades profissionais da área da comunicação, em termos tecnológicos, organizacionais e laborais, consideramos como prioridade o resgatar de memórias e vestígios plurais que tendem a ser ignorados e esquecidos, nas fontes oficiais e nos registos institucionais sobre a área da comunicação social. Optámos aqui por tomar a atividade da comunicação social como um todo, rejeitando a perspectiva de uma análise parcelar, por sector ou por suporte tecnológico, colocando as várias áreas ligadas aos media como um conjunto ocupacional (onde muitas trajetórias de vida cruzam diferentes áreas). Procuraremos diversificar as “ vozes” que “contam”, a nossa preocupação recairá na inclusão dos diversos grupos de profissionais, do diretor de informação de um canal de televisão, ao tipógrafo, ao gestor de comunicação, ao jornalista, ao account em comunicação, ao diretor do serviço ao cliente, ao revisor, ao assessor de imprensa, ao operador de imagem, ao copyright, ao fotógrafo, ao regente de estúdios, ao sonoplasta, etc. O caracter interdisciplinar é assegurado por uma equipa que 21 investigadores, de várias instituições do ensino superior, de Portugal e do Brasil. O nosso objectivo é alargar o projeto a outras instituições académicas e associações profissionais da área da comunicação. O projeto está numa fase de arranque, para a qual obteve financiamento em Dezembro de 2017, no Concurso Anual para Projectos de Investigação, Desenvolvimento, Inovação e Criação Artística do IPL-2016.Este trabalho insere-se no projeto de Investigação, Desenvolvimento, Inovação e Criação Artística do Instituto Politécnico de Lisboa (IDI&CA do IPL – 2016) com a referência IPL/2016/AMOPC_ESCS.N/

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research