New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response

Evolution of spray and aerosol from respiratory releases: theoretical estimates for insight on viral transmission

By modelling the evaporation and settling of droplets emitted during respiratory releases and using previous measurements of droplet size distributions and SARS-CoV-2 viral load, estimates of the evolution of the liquid mass and the number of viral copies suspended were performed as a function of time from the release. The settling times of a droplet cloud and its suspended viral dose are significantly affected by the droplet composition. The aerosol (defined as droplets smaller than 5 μm) resulting from 30 s of continued speech has O(1 h) settling time and a viable viral dose an order-of-magnitude higher than in a short cough. The time-of-flight to reach 2 m is only a few seconds resulting in a viral dose above the minimum required for infection, implying that physical distancing in the absence of ventilation is not sufficient to provide safety for long exposure times. The suspended aerosol emitted by continuous speaking for 1 h in a poorly ventilated room gives 0.1–11% infection risk for initial viral loads of 108–1010 copies ml−ll, respectively, decreasing to 0.03–3% for 10 air changes per hour by ventilation. The present results provide quantitative estimates useful for the development of physical distancing and ventilation controls

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases

Milena Fronza Broering,1,2 Pedro Leonidas Oseliero Filho,3,4 Pâmela Pacassa Borges,1 Luis Carlos Cides da Silva,3 Marcos Camargo Knirsch,5 Luana Filippi Xavier,1 Pablo Scharf,1 Silvana Sandri,1 Marco Antonio Stephano,5 Fernando Anselmo de Oliveira,6 Ibrahim M Sayed,2 Lionel Fernel Gamarra,6 Soumita Das,2 Márcia CA Fantini,3 Sandra HP Farsky1 1Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil; 2Department of Biomedical and Nutritional Sciences, University of Massachusetts, Lowell, MA, USA; 3Department of Applied Physics, Physics Institute, University of Sao Paulo, São Paulo, Brazil; 4Materials Innovation Factory, University of Liverpool, Liverpool, MSY, UK; 5Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil; 6Instituto do Cérebro, Instituto Israelita de Ensino e Pesquisa, Sociedade Beneficente Israelita Brasileira Hospital Albert Einstein, São Paulo, SP, BrazilCorrespondence: Sandra HP Farsky, Email [email protected]: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology.Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut.Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 μg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis.Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results.Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy. Keywords: annexin A1, SBA-15, oral route, tissue recovery, inflammatio

Acuidade olfatória e qualidade de vida após a laringectomia total

RESUMO: Objetivo: identificar a prevalência e os fatores associados às alterações do olfato e descrever a qualidade de vida após a laringectomia total. Métodos: estudo transversal para avaliar a acuidade olfatória e a qualidade de vida de laringectomizados totais no Instituto Nacional de Câncer por meio da aplicação do Teste de Identificação do Olfato da Universidade da Pensilvânia, do Questionário de Qualidade de Vida da Universidade de Washington e do Questionário sobre a Acuidade Olfatória Pré-Reabilitação do Olfato. Resultados: foram avaliados 48 laringectomizados totais, sendo 39 do sexo masculino e 9 do sexo feminino, com idade média de 62 anos e tempo médio de 5,6 anos desde a laringectomia total. No Teste de Identificação do Olfato a pontuação média foi de 17,9. De acordo com a classificação do olfato no teste, a maioria dos participantes apresentou algum grau de alteração, sendo que apenas 2 indivíduos tiveram o olfato considerado dentro da normalidade. No Questionário de Qualidade de vida da Universidade de Washington, o escore composto foi 80,47. Os domínios que apresentaram as médias de pontos mais baixas foram paladar, saliva e fala. No questionário sobre a acuidade olfatória pré-reabilitação do olfato, a maioria dos participantes consideraram seu olfato de ruim a razoável. 21 indivíduos relataram apresentar algum grau de dificuldade em suas atividades de vida diária em decorrência de alterações do olfato. Conclusão: os laringectomizados totais apresentaram alta prevalência de alterações do olfato, com comprometimentos relacionados às suas atividades de vida diária