Live Visualization of Hemagglutinin Dynamics During Infection by Using a Novel Reporter Influenza A Virus

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing

O lodo ativo da planta de uma indústria de petróleo é constituído por uma microbiota ainda a ser identificada

The active sludge process is one of the most-used techniques for the biodegradation of organic compounds present in effluents from an assortment of wastewaters. This study investigated the bacterial community structure of a petroleum industry’s activated sludge and its physical and chemical parameters using high-throughput sequencing. Samples were collected over one year: autumn 2015 (C1), winter 2015 (C2), spring 2015 (C3), and summer 2016 (C4). Total DNA was extracted, and the primers targeting the V4 region of the 16S rRNA gene were used for amplicon sequencing. The majority of the detected microorganisms were considered rare microbiota, presenting a relative abundance below 1% of the total sequences. All of the sequences were classified at the phylum level, and up to 55% of the ASVs (Amplicon Sequence Variants) were associated with known bacterial genera. Proteobacteria was the most abundant phylum in three seasons, while the phylum Armatimonadota dominated in one season. The genus Hyphomicrobium was the most abundant in autumn, winter and summer, and an ASV belonging to the family Fimbriimonadaceae was the most abundant in the spring. Canonical Correspondence Analysis showed that physicochemical parameters of SS, SD and TSS are correlated, as well as ammoniacal nitrogen. Sample C3 presented the highest values of COD, AN and solids (SS, SD and TSS). The highest COD, AN, and solids values are correlated to the high frequency of the phylum Armatimonadota in C3.O processo de lodo ativo é uma das técnicas mais utilizadas para biodegradação de compostos orgânicos presentes nos efluentes de uma variedade de águas residuais. A estrutura da comunidade bacteriana do lodo ativado de uma indústria de petróleo e sua relação com parâmetros físicos e químicos foram investigadas por meio de sequenciamento de alto rendimento. As amostras foram coletadas durante um período de um ano: outono de 2015 (C1), inverno de 2015 (C2), primavera de 2015 (C3) e verão de 2016 (C4). O DNA total foi extraído e para amplificação foram utilizados primers específicos para região V4 do gene 16S rRNA. A maioria dos microrganismos detectados foi considerada microbiota rara, apresentando abundância relativa abaixo de 1% do total de sequências. Em geral, quase a totalidade das sequências (99,9%) foi classificada em nível de filo, mas apenas algumas ASVs (23,7%) foram associadas a gênero bacteriano conhecido. As proteobactérias foram o filo mais abundante em três das estações, enquanto o filo Armatimonadota dominou em uma estação. O gênero Hyphomicrobium foi o gênero mais abundante no outono, inverno e verão, e uma ASV pertencente à família Fimbriimonadaceae (filo Armatimonadetes) foi o microrganismo mais abundante na primavera. A Análise de Correspondência Canônica (CCA) indica uma diferença consistente da comunidade bacteriana da primavera quando comparada com amostras de outras estações. Os resultados mostram uma correlação entre o filo Armatimonadota e a alta concentração de DQO, NA e sólidos

Pesquisa de anticorpos contra o vírus da imunodeficiência humana tipos 1 e 2 em amostras de sangue seco coletadas em papel filtro

Human Immunodeficiency Vírus Type 1 and 2 antibodies detection was performed in 457 dried whole blood spots samples (S&S 903). Q-Preven HIV 1+2 was the screening test used. The results were compared with the gold standard serum tests by ELISA (Cobas Core e Axsym HIV1/2 gO) and imunofluorescence was the definitive confirmatory test. The samples were obtained from the Hospital Nossa Senhora da Conceição in Porto Alegre, RS - Brazil, through whole blood transfer to filter paper card and sent to Caxias do Sul, RS - Brazil where the tests were performed. The dried whole blood spot stability was evaluated with two different panels. The first one was composed of five negative and five positive samples stored at room temperature, 4 ºC, -20 ºC and -70 ºC, while the second was composed of two negative and three positive samples stored at 37 ºC (humidityForam realizados 457 testes para detectar anticorpos contra o Vírus da Imunodeficiência Humana tipos 1 e 2, em amostras de sangue total seco coletadas em papel filtro (S&S 903), com o teste de triagem Q-Preven HIV 1+2, comparando-se com os resultados dos testes de triagem no soro (Cobas Core e Axsym HIV1/2 gO), sendo a imunofluorescência indireta o teste confirmatório. As amostras foram obtidas no Hospital Conceição em Porto Alegre, pela transferência de sangue total para cartão de papel filtro e encaminhadas para Caxias do Sul para a realização dos testes. Foi analisada a estabilidade da amostra em papel filtro com a utilização de dois painéis: o primeiro com cinco amostras negativas e cinco positivas armazenadas por seis semanas à temperatura ambiente, 4 ºC, -20 ºC e -70 ºC; o segundo com duas negativas e três positivas armazenadas por seis semanas com avaliações semanais a 37 ºC (umidad

Efficiency of probiotic traits in plant inoculation is determined by environmental constrains

Management of the soil microbial community to increase crop productivity is one of the main challenges of modern agriculture, and bacterial inoculants can help to overcome this challenge. In this work, two plant growth-promoting (PGP) bacteria were evaluated under contrasting soil conditions, in order to test a rhizosphere ecology model. This model states that plants select for phosphate (P) solubilizers in poor nutrient soils and Indolic Compounds (ICs) producers in rich nutrient soils. Rice plants were single- or co-inoculated with strains of Burkholderia and Enterobacter genera in clayey and sandy soils. Diversity gradients were generated in each soil type using the dilution-to-extinction approach. Inoculant survival, colonization, and effect on plant biomass were evaluated, besides P solubilization and ICs production from both rhizospheric and endophytic bacterial communities. The PGP efficiency of the Burkholderia strain was highest in the sandy soil, which had the highest bacterial P solubilization potential, whereas the PGP efficiency of the Enterobacter strain was highest in the clayey soil, which had the highest bacterial ICs production potential. These behaviors occurred as hypothesized by the model, which can be useful for PGPB testing and bioprospection. We highlight a strong dependency of the Enterobacter strain on the diversity level as the most critical factor affecting PGP efficiency, possibly related to the increased influence of keystone taxa in lower diversity as indicated by network analysis

Fecal eukaryotic community of wild young South American (Arctocephalus australis) and Subantarctic fur seals (Arctocephalus tropicalis)

Eukaryotic microbes that reside in the mammalian gut have an important role in maintaining metabolism, digesting nutrients, and regulating the immune system. Therefore, changes in the microbial composition of the gut may generate adverse impacts on animal health. Using high-throughput sequencing, the present study examined the fecal eukaryotic community of wild young South American (Arctocephalus australis) (n = 2) and Subantarctic fur seals (Arctocephalus tropicalis) (n = 3). The results indicated there was a distinct and diverse eukaryotic community in the fecal samples of young wild fur seals. Perhaps based on the migratory habits of certain species and the difficulty in obtaining samples, the microbiota of wild animals is poorly understood. This work reports a number of phyla and classes of microorganisms never noticed in the fecal samples of wild fur seals before and provide insight into the fecal eukaryotic community of wild young South American and Subantarctic fur seals

Enrichment of sulphate-reducers and depletion of butyrate-producers may be hyperglycaemia signatures in the diabetic oral microbiome

Objectives: This study aimed to investigate oral microbial signatures associated with hyperglycaemia, by correlating the oral microbiome with three glycaemic markers. Potential association between clinical parameters and oral bacterial taxa that could be modulating the hyperglycaemic microbiome was also explored. Methods: Twenty-three individuals diagnosed with type 2 Diabetes Mellitus (T2D) and presenting periodontitis were included, as well as 25 systemically and periodontally healthy ones. Fasting blood glucose, glycated haemoglobin, salivary glucose, periodontitis classification, caries experience and activity and salivary pH were evaluated. The V4 region of the 16S rRNA gene was amplified from total salivary DNA, and amplicons were sequenced (Illumina MiSeq). Results: Hyperglycaemia was correlated with proportions of Treponema, Desulfobulbus, Phocaiecola and Saccharimonadaceae. Desulfobulbus was ubiquitous and the most enriched organism in T2D individuals (log2FC = 4). The Firmicutes/Bacteroidetes ratio was higher at alkali salivary pH than acidic pH. In the network analysis, Desulfobulbus was clustered in a negative association with caries-associated and butyrate-producing bacteria. Conclusion: The salivary microbiome is shaped by systemic hyperglycaemia, as well as changes in the salivary pH, which may be linked to local hyperglycaemia. The enrichment of predictive biomarkers of gut dysbiosis in the salivary microbiome can reflect its capacity for impairment of hyperglycaemia

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Live Visualization of Hemagglutinin Dynamics During Infection by Using a Novel Reporter Influenza A Virus

Live visualization of influenza A virus (IAV) structural proteins during viral infection in cells is highly sought objective to study different aspects of the viral replication cycle. To achieve this, we engineered an IAV to express a Tetra Cysteine tag (TC tag) from hemagglutinin (HA), which allows intracellular labeling of the engineered HA protein with biarsenic dyes and subsequent fluorescence detection. Using such constructs, we rescued a recombinant IAV with TC tag inserted in HA, in A/Puerto Rico/8/1934(H1N1) background (HA-TC). This recombinant HA-TC tag reporter IAV was replication-competent; however, as compared to wild type PR8 IAV, it was attenuated in multicycle replication. We confirmed expression of TC tag and biarsenical labeling of HA by immunofluorescence assay in cells infected with an HA-TC tag reporter IAV. Further, we used this reporter virus to visualize HA expression and translocation in IAV infected cells by live confocal imaging. We also tested the utility of the HA-TC IAV in testing chemical inhibitors of the HA translocation. Overall, HA-TC IAV is a versatile tool that will be useful for studying viral life cycle events, virus-host interactions, and anti-viral testing