Characterization and applicability of a novel physiologically relevant 3D-tetraculture bronchial model for in vitro assessment of respiratory sensitization.

Chemical Respiratory Allergy (CRA) is triggered after exposure to Low Molecular Weight (LMW) sensitizers and manifests clinically as asthma and rhinitis. From a risk/toxicity assessment point of view, there are few methods, none of them validated, for evaluating the respiratory sensitization potential of chemicals once the in vivo-based models usually employed for inhalation toxicity addressment do not comprise allergenicity endpoints specifically. Based on that, we developed, characterized, and evaluated the applicability of a 3D-tetraculture airway model reconstructed with bronchial epithelial, fibroblasts, endothelial and monocytic cell lines. Moreover, we exposed the tissue to maleic anhydride (MA) aerosols to challenge the model and subsequently assessed inflammatory and functional aspects of the tissue. The reconstructed tissue presented phenotypic biomarkers compatible with human bronchial epithelium, and MA aerosol exposure triggered an increased IL-8 and IL-6 production, reactive oxygen species (ROS) formation, and apoptosis of epithelial cells. Besides, augmented IL-8 production by monocytic cells was also found, correlating with dendritic cell activation within the co-culture model after MA exposure. Our results demonstrated that the 3D-tetraculture bronchial model presents hallmarks related to human airways' structure and function. Additionally, exposure to a respiratory sensitizer induced inflammatory and functional alterations in the reconstructed tissue, rendering it a valuable tool for exploring the mechanistic framework of chemically induced respiratory sensitization.

BADASS: BActeriocin-Diversity ASsessment Software.

BACKGROUND: Bacteriocins are defined as thermolabile peptides produced by bacteria with biological activity against taxonomically related species. These antimicrobial peptides have a wide application including disease treatment, food conservation, and probiotics. However, even with a large industrial and biotechnological application potential, these peptides are still poorly studied and explored. BADASS is software with a user-friendly graphical interface applied to the search and analysis of bacteriocin diversity in whole-metagenome shotgun sequencing data. RESULTS: The search for bacteriocin sequences is performed with tools such as BLAST or DIAMOND using the BAGEL4 database as a reference. The putative bacteriocin sequences identified are used to determine the abundance and richness of the three classes of bacteriocins. Abundance is calculated by comparing the reads identified as bacteriocins to the reads identified as 16S rRNA gene using SILVA database as a reference. BADASS has a complete pipeline that starts with the quality assessment of the raw data. At the end of the analysis, BADASS generates several plots of richness and abundance automatically as well as tabular files containing information about the main bacteriocins detected. The user is able to change the main parameters of the analysis in the graphical interface. To demonstrate how the software works, we used four datasets from WMS studies using default parameters. Lantibiotics were the most abundant bacteriocins in the four datasets. This class of bacteriocin is commonly produced by Streptomyces sp. CONCLUSIONS: With a user-friendly graphical interface and a complete pipeline, BADASS proved to be a powerful tool for prospecting bacteriocin sequences in Whole-Metagenome Shotgun Sequencing (WMS) data. This tool is publicly available at https://sourceforge.net/projects/badass/ .

Development of a novel ex vivo model for chemical ocular toxicity assessment and its applicability for hair straightening products.

This study developed an air-liquid interface (ALI) corneal model using explants bovine eyes for ocular toxicity assessment of ten chemicals and seven hair straightening mixtures. It was successfully maintained physiologically viable and normal for six days. Both eye damage (GHS cat. 1) and irritating (GHS cat. 2) chemicals induced corneal injury in our model. However, cat. 2 irritants triggered moderate damage when compared to cat. 1 agents, which induced a marked cytotoxicity profile. The mixtures were also able to trigger viability reduction associated with histopathological changes in the corneal tissues, especially when the exposure was via aerosol particles. Thus, the chemical exposure microenvironment simulation seemed to provide more reliable toxicological data. Moreover, mixture-induced corneal damage correlated with increased ROS levels, suggesting a close correlation between tissue death and oxidative stress. Besides mixtures showing the potential to induce moderate/mild ocular toxicity, we could verify that the corneal tissue damage showed reversibility due to the recovery from the injury after exposure to some of the mixtures. Hence, our ex vivo corneal model seems to be a simple and cost-effective approach for future studies related to further investigating the reversibility of damage in the cornea triggered by chemicals and their mixtures.

Innovative strategy based on mechanisms to substitute animal testing for ocular toxicity assessment of agrochemical formulations market in Brazil.

Considering the successful employment of alternative methods for eye toxicity assessment of products for regulatory purposes, and the recent advances in Brazilian legislative scenario, which adopted the UN GHS classification system for agrochemical formulations toxicity assessment, there is an emerging demand for strategies that allow the evaluation of such products. Based on this, the present study aimed to address the applicability of a mechanistic-based defined approach for eye toxicity assessment of agrochemical formulations. It was investigated the opacity/permeability, depth and location of corneal injury in bovine cornea, and vascular events in chorioallantoic membrane induced for different Brazilian agrochemicals using a Sequential Testing Strategy (STS). Cytotoxicity induced by the agrochemical formulations was evaluated by Short Time exposure (STE) (OECD TG 491) assay (step 1), corneal injury was investigated by standard Bovine Corneal Opacity and Permeability (BCOP) (OECD TG 437) followed by histopathological evaluation (step 2), and Hen Chorionic-allantoic Membrane test (HET-CAM) was used to evaluate vascular injury (step 3). The results demonstrated that the proposed defined approach enabled a classification corresponding UN GHS classification of agrochemical formulations while minimizing the use of live animals. Therefore, this approach may be useful for categorization of agrochemicals in Brazil according to the new regulatory scenario.

Characterization and Cytotoxicity Assessment of the Lipophilic Fractions of Different Morphological Parts of Acacia dealbata.

Acacia dealbata biomass, either from forest exploitation or from the management of invasive species, can be a strategic topic, namely as a source of high-value compounds. In this sense, the present study aimed at the detailed characterization of the lipophilic components of different morphological parts of A. dealbata and the evaluation of their cytotoxicity in cells representative of different mammals' tissues. The chemical composition of lipophilic extracts from A. dealbata bark, wood and leaves was evaluated using gas chromatography-mass spectrometry (GC-MS). Terpenic compounds (representing 50.2%-68.4% of the total bark and leaves extracts, respectively) and sterols (60.5% of the total wood extract) were the main components of these extracts. Other constituents, such as fatty acids, long-chain aliphatic alcohols, monoglycerides, and aromatic compounds were also detected in the studied extracts. All the extracts showed low or no cytotoxicity in the different cells tested, demonstrating their safety profile and highlighting their potential to be used in nutraceutical or pharmaceutical applications. This study is therefore an important contribution to the valorization of A. dealbata, demonstrating the potential of this species as a source of high value lipophilic compounds.

A new corneal epithelial biomimetic 3D model for in vitro eye toxicity assessment: Development, characterization and applicability.

In vitro eye toxicity assessment using reconstructed corneal epithelial models has emerged highlighting its applicability domain for Classification and Labeling of products and chemicals. However, due to bureaucratic issues, such models are not commercially available in Brazil and Latin America. In this work, we developed, characterized and evaluated the applicability of a new corneal epithelial biomimetic model using a cell lineage for in vitro eye toxicity assessment. The reconstructed tissue was obtained through the cultivation of HaCaT cells in an air-liquid interface, which presented morphology and biomarkers expression such as cytokeratin, CD44, and Ki-67 similar to human tissue. Furthermore, tissue viability was evaluated after exposure of the epithelial model to isolated chemicals from different Globally Harmonized System (GHS) eye irritation categories, and it has been demonstrated to be a suitable endpoint for classification of test materials, allowing discrimination between irritant and non-irritant chemicals. Furthermore, the model showed suitability for testing "real-life mixtures", once it identified irritant products between the analyzed eyebrow henna samples commercially labeled as non-irritants. This reproducible and low-cost epithelial corneal model presents features very important for Brazil and South America for R&D&I with no unnecessary animal experimentation.

Proposal for an ecofriendly and economic strategy for efficient radioiodination of coumarin derivatives.

Combination of the calculation of reactivity descriptors and the cold iodine test for some coumarin derivatives was used in order to optimize the radioiodination reaction. The strongly nucleophilic predicted coumarins were subjected to the action of cold iodine. With two coumarins substituted at 3 by the 2-hydroxybenzoyl group, iodination did not occur but a product of intramolecular heterocyclization was obtained. This strategy is useful for economic and environmentally friendly radioiodination.

Mechanistic-based non-animal assessment of eye toxicity: Inflammatory profile of human keratinocytes cells after exposure to eye damage/irritant agents.

Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24 h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24 h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1ß, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1ß production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1ß were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1ß measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.

Produtivismo na saúde: desafios do SUS na invenção da gestão democrática / Health in productivism: challenges of SUS in the invention of democratic management

A Associação Brasileira de Editoras Universitárias lançou o livro Produtivismo na Saúde – Desafios do SUS na invenção da Gestão democrática. Fruto de uma pesquisa desenvolvida conjuntamente por quatro grupos de pesquisas acadêmicas (três da Universidade Federal de Pernambuco e um do Centro de Pesquisas Aggeu Magalhães/Fiocruz Pernambuco), a obra busca aprofundar criticamente o tema do produtivismo na saúde a partir de diferentes abordagens: sociais, jurídicas, da saúde coletiva e de saberes e práticas de saúde. Com 231 páginas, o livro é dividido em quatro capítulos que abordam os temas: Gestão em saúde e produtivismo; Humanização, parto e nascimento; Sofrimento, subjetividade e medicalização; e por fim, Práticas antiutilitaristas e integrativas em saúde. Para Islândia Sousa, pesquisadora da Fiocruz PE e uma das coordenadoras da publicação, a obra possui ineditismo por discutir de modo interdisciplinar os efeitos negativos do modelo econômico no campo da saúde, o que favoreceu um olhar ampliado acerca do tema, e também apresenta experiências que visam minimizar os estes efeitos...

Ion Torrent-based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high-performance liquid chromatography a promising rRNA depletion method.

Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.

Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

BACKGROUND: Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. FINDINGS: We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. CONCLUSIONS: Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

Avaliação da experiência de cárie e necessidade de tratamento de pré-escolares de 5 e 6 anos das redes pública e privada do município de Porto Velho, Rondônia / Evaluation of caries experience and treatment needs of children of 5 and 6 age of public and private schools in the city of porto velho, rondônia

O objetivo do estudo foi o de comparar a prevalência de cárie de pré-escolares do município de Porto Velho.Foram examinadas 227 crianças, de 5 e 6 anos, sendo 85 (37,4%) de uma escola privada (A), 79 (34,8%) deuma escola municipal localizada no centro da cidade (B), e 63 (27,8%) de uma escola municipal da periferia(C). Os resultados foram submetidos à análise de variância e ao teste t, com diferença estatisticamente significante(p <= 0,05), quando comparados os índices das três escolas (A<=B<=C). A quantidade de participanteslivres de cárie na Escola A (77,65%) foi maior do que nas escolas B e C (20% e 2,35%, respectivamente).Restauração de uma superfície foi a necessidade de tratamento mais encontrada nas Escolas A e C (45,45%e 40,66%). Entretanto, na Escola B, a restauração de duas superfícies foi a mais observada (50,99%). Comrelação aos componentes do índice ceo-d, tanto nas escolas públicas (B=76,4% e C=92,83%) quanto naprivada (61,79%), o componente c (cariado) foi o de maior prevalência. Os resultados sugerem que o nívelsocioeconômico das crianças pesquisadas, a partir da escola em que estudavam e da localização desta teminfluência nos índices de cárie, indicando a importância de se planejarem estratégias adequadas para cada grupo da população.

The aim of this study was to compare the level of tooth decay of chldren from the city Porto Velho, 227 children on age 5 and 6 were evaluated, been 85 (37,4%) of private school (A), 79 (34,8%) of a public school that is located in the center of Porto Velho (B) ande also 63 (27,8%) of a public school that is located in periphery of the city (C). The results were submitted to a variance analysis and to t test, with significant statically difference (p<=0,05) when compared to the index of the three schools (A<=B<=C). The amount of tooth decay free patients in school A (77,65%) was bigger than school B and C (20% and 2,35%), respectively). Restorations of one face of the teeth were the treatment needed most found in school A and C (45,5% and 40,66%), however in school B restorations of the teeth's faces of the teeth were the most seen (50,59%). In relation to the compound of ceo-d index, as in public school (B=76,4% and C=92,83%), as in the private school (61,79%), the compound C (tooth decay) was the most prevalent. The results suggest that socioeconomic level of the children investigated, from the school that they study and its location, has an influence in the tooth decav index. Indicating the importance to plan appropriate strategies for each population group.

Spectroscopic and electrochemical studies of cocaine-opioid interactions.

The drugs of abuse cocaine (C), heroin (H), and morphine (M) have been studied to enable understanding of the occurrence of cocaine-opioid interactions at a molecular level. Electrochemical, Raman, and NMR studies of the free drugs and their mixtures were used to study drug-drug interactions. The results were analyzed using data obtained from quantum-mechanical calculations. For the cocaine-morphine mixture (C-MH), formation of a binary complex was detected; this involved the 3-phenolic group and the heterocyclic oxygen of morphine and the carbonyl oxygen and the methyl protons of cocaine's methyl ester group. NMR studies conducted simultaneously also revealed C-MH binding geometry consistent with theoretical predictions and with electrochemical and vibrational spectroscopy results. These results provide evidence for the occurrence of a cocaine-morphine interaction, both in the solid state and in solution, particularly for the hydrochloride form. A slight interaction, in solution, was also detected by NMR for the cocaine-heroin mixture.