Endocytosis of DNA-Hsp65 Alters the pH of the Late Endosome/Lysosome and Interferes with Antigen Presentation

BACKGROUND: Experimental models using DNA vaccine has shown that this vaccine is efficient in generating humoral and cellular immune responses to a wide variety of DNA-derived antigens. Despite the progress in DNA vaccine development, the intracellular transport and fate of naked plasmid DNA in eukaryotic cells is poorly understood, and need to be clarified in order to facilitate the development of novel vectors and vaccine strategies. METHODOLOGY AND PRINCIPAL FINDINGS: Using confocal microscopy, we have demonstrated for the first time that after plasmid DNA uptake an inhibition of the acidification of the lysosomal compartment occurs. This lack of acidification impaired antigen presentation to CD4 T cells, but did not alter the recruitment of MyD88. The recruitment of Rab 5 and Lamp I were also altered since we were not able to co-localize plasmid DNA with Rab 5 and Lamp I in early endosomes and late endosomes/lysosomes, respectively. Furthermore, we observed that the DNA capture process in macrophages was by clathrin-mediated endocytosis. In addition, we observed that plasmid DNA remains in vesicles until it is in a juxtanuclear location, suggesting that the plasmid does not escape into the cytoplasmic compartment. CONCLUSIONS AND SIGNIFICANCE: Taken together our data suggests a novel mechanism involved in the intracellular trafficking of plasmid DNA, and opens new possibilities for the use of lower doses of plasmid DNA to regulate the immune response

The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Visceral Leishmaniasis Urbanization in the Brazilian Amazon Is Supported by Significantly Higher Infection Transmission Rates Than in Rural Area

This was an open cohort prospective study (2016–2018) that analyzed the prevalence and incidence rates of human Leishmania (L.) infantum chagasi-infection and the evolution of their clinical-immunological profiles in distinct urban and rural scenarios of American visceral leishmaniasis (AVL) in Pará State, in the Brazilian Amazon. These infection profiles were based on species-specific DTH/IFAT-IgG assays and clinical evaluation of infected individuals, comprising five profiles: three asymptomatic, Asymptomatic Infection [AI], Subclinical Resistant Infection [SRI], and Indeterminate Initial Infection [III]; and two symptomatic, Subclinical Oligosymptomatic Infection [SOI] and Symptomatic Infection [SI = AVL]. The two distinct scenarios (900 km away) were the urban area of Conceição do Araguaia municipality and the rural area of Bujaru municipality in the southeast and northeast of Pará State. Human populations were chosen based on a simple convenience sampling design (5–10% in each setting), with 1723 individuals (5.3%) of the population (32,464) in the urban area and 1568 individuals (8.9%) of the population (17,596) in the rural one. A serological survey (IFAT-IgG) of canine infection was also performed in both scenarios: 195 dogs in the urban area and 381 in the rural one. Prevalence and incidence rates of human infection were higher in the urban area (20.3% and 13.6/100 person-years [py]) than in the rural setting (14.1% and 6.8/100-py). The AI profile was the most prevalent and incident in both urban (13.4% and 8.1/100-py) and rural (8.3% and 4.2/100-py) scenarios, but with higher rates in the former. An III profile case evolved to SOI profile after four weeks of incubation and another to SI (=AVL) after six. The prevalence of canine infection in an urban setting (39.2%) was also higher (p < 0.05) than that (32%) in the rural zone. AVL urbanization in Pará State, in the Brazilian Amazon, has led to infection rates significantly higher than those in rural sites, requiring more intense control measures

Estudo de base populacional dos fatores associados à incapacidade funcional entre idosos na Região Metropolitana de Belo Horizonte, Minas Gerais, Brasil A population-based study on factors associated with functional disability among older adults in the Great Metropolitan Belo Horizonte, Minas Gerais State, Brazil

O objetivo deste trabalho foi determinar os fatores associados à incapacidade funcional entre idosos na Região Metropolitana de Belo Horizonte, Minas Gerais, Brasil. O modelo teórico adotado foi baseado em características predisponentes (sócio-demográficas), fatores extra-individuais (apoio social, uso de serviços de saúde) e intra-individuais (condições de saúde). Participaram do estudo 1.786 idosos (> 60 anos) selecionados por meio de amostra probabilística. A variável dependente foi a incapacidade funcional, definida como incapacidade leve ou moderada (alguma dificuldade) e grave (total dependência) para realizar atividades da vida diária. A prevalência da incapacidade foi de 16% (8% leve e 8% grave). Idade e pior auto-avaliação da saúde apresentaram associações positivas e independentes com ambos os níveis de incapacidade. Hipertensão e artrite apresentaram associações com incapacidade leve ou moderada, enquanto diabetes e acidente vascular cerebral apresentaram associações com incapacidade grave. Associação negativa com incapacidade grave foi observada para visita de amigos nos últimos trinta dias. Esses resultados mostram que as condições crônicas associadas à incapacidade na população estudada são passíveis de prevenção e que o apoio social externo à família é menor em idosos com incapacidade grave.<br>This study assessed factors associated with functional disability in old age in Greater Metropolitan Belo Horizonte, Minas Gerais State, Brazil. The theoretical model was based on predisposing (socio-demographic), extra-individual (social support, use of health services), and intra-individual factors (health conditions). The study was conducted in a probabilistic sample of 1,786 subjects aged > 60 years. The dependent variable was disability defined as mild (some difficulty) or severe (total dependence) in performing at least one basic activity of daily living. Prevalence of disability was 16% (8% mild; 8% severe). Age and worse self-rated health were independently and positively associated with mild and severe disability. Self-reported hypertension and arthritis were associated with mild disability, while diabetes and stroke were associated with severe disability. Severe disability was independently and negatively associated with number of visits by friends in the previous 30 days. According to the results, chronic conditions associated with disability in the study population are preventable, and severely disabled elderly had less extra-family social support

Monitoramento atmosférico passivo de SO2, NO2 e O3 em áreas urbanas e de influência industrial como prática de química ambiental para alunos de graduação Atmospheric passive monitoring of SO2, NO2 and O3 in urban areas and in those under industrial influence as an environmental chemistry experiment for undergraduate students

<abstract language="eng">This study is a result of undergraduate student participation in the Environmental Chemistry discipline provided by the Chemistry Institute/UFBA. The students were involved in the development of passive samplers, a project of the LAQUAM (Environmental Analytical Chemistry Laboratory). The students' residences and other neighborhoods were used to create a passive sampling network, allowing the measurement of atmospheric levels of pollutants in urban areas and in those under industrial influence. The assembly of the passive samplers, including impregnation of filters and chemical analysis were part of the students' practice tasks. The results were analyzed taking into consideration the Brazilian legislation