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The prevalence of the highly pathogenic avian influenza virus H5N1 in wild birds that migrate all over the world has resulted in the dissemination of this virus across Asia, Europe, Africa, North and South America, the Arctic continent, and Antarctica. So far, H5N1 clade 2.3.4.4.b has reached an almost global distribution, with the exception of Australia and New Zealand for autochthonous cases. H5N1 clade 2.3.4.4.b, derived from the broad-host-range A/Goose/Guangdong/1/96 (H5N1) lineage, has evolved, adapted, and spread to species other than birds, with potential mammal-to-mammal transmission. Many public health agencies consider H5N1 influenza a real pandemic threat. In this sense, we analyzed H5N1 hemagglutinin sequences from recent outbreaks in animals, clinical samples, antigenic prototypes of candidate vaccine viruses, and licensed human vaccines for H5N1 with the aim of shedding light on the development of an H5N1 vaccine suitable for a pandemic response, should one occur in the near future.

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Maternal Zika virus (ZIKV) infection during pregnancy has been associated with severe intrauterine growth restriction (IUGR), placental damage, metabolism disturbances, and newborn neurological abnormalities. Here, we investigated the impact of maternal ZIKV infection on placental nutrient transporters and nutrient-sensitive pathways. Immunocompetent (C57BL/6) mice were injected with Low (103 PFU-ZIKVPE243) or High (5 × 107 PFU-ZIKVPE243) ZIKV titers at gestational day (GD) 12.5, and tissue was collected at GD18.5 (term). Fetal-placental growth was impaired in male fetuses, which exhibited higher placental expression of the ZIKV infective marker, eukaryotic translation initiation factor 2 (eIF2α), but lower levels of phospho-eIF2α. There were no differences in fetal-placental growth in female fetuses, which exhibited no significant alterations in placental ZIKV infective markers. Furthermore, ZIKV promoted increased expression of glucose transporter type 1 (Slc2a1/Glut1) and decreased levels of glucose-6-phosphate in female placentae, with no differences in amino acid transport potential. In contrast, ZIKV did not impact glucose transporters in male placentae but downregulated sodium-coupled neutral amino acid 2 (Snat2) transporter expression. We also observed sex-dependent differences in the hexosamine biosynthesis pathway (HBP) and O-GlcNAcylation in ZIKV-infected pregnancies, showing that ZIKV can disturb placental nutrient sensing. Our findings highlight molecular alterations in the placenta caused by maternal ZIKV infection, shedding light on nutrient transport, sensing, and availability. Our results also suggest that female and male placentae employ distinct coping mechanisms in response to ZIKV-induced metabolic changes, providing insights into therapeutic approaches for congenital Zika syndrome.

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The prevalence of the highly pathogenic avian influenza virus H5N1 in wild birds that migrate all over the world has resulted in the dissemination of this virus across Asia, Europe, Africa, North and South America, the Arctic continent, and Antarctica. So far, H5N1 clade 2.3.4.4.b has reached an almost global distribution, with the exception of Australia and New Zealand for autochthonous cases. H5N1 clade 2.3.4.4.b, derived from the broad-host-range A/Goose/Guangdong/1/96 (H5N1) lineage, has evolved, adapted, and spread to species other than birds, with potential mammal-to-mammal transmission. Many public health agencies consider H5N1 influenza a real pandemic threat. In this sense, we analyzed H5N1 hemagglutinin sequences from recent outbreaks in animals, clinical samples, antigenic prototypes of candidate vaccine viruses, and licensed human vaccines for H5N1 with the aim of shedding light on the development of an H5N1 vaccine suitable for a pandemic response, should one occur in the near future.

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Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

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Abstract Introduction Myringotomy and ear tube placement (MTP) is the surgical treatment for otitis media with effusion (OME), and it is the most common surgery performed in children. Several guidelines have been developed to assist in the care of patients who become candidates for MTP. Objectives To evaluate the practice of Brazilian otorhinolarynogologists when performing MTP according to the years of clinical experience. Secondarily, we also want to assess if their practice regarding MTP varied according to the percentage of children treated and the location of their practice. Methods A 30-question survey was sent to otolaryngologists affiliated with the Brazilian Academy of Pediatric Otorhinolaryngology (Academia Brasileira de Otorrinolaringologia Pediátrica, ABOPe, in Portuguese) and/or the Scientific Department of Otorhinolaryngology of the Brazilian Society of Pediatrics (Sociedade Brasileira de Pediatria, SBP, in Portuguese). The questions included were carefully chosen to provide a profile about the practices adopted in the pre-, peri- and postoperative periods of MTP. Results The questionnaire was sent to 208 otolaryngologists, and there were 124 (59.6%) respondents. Of those, 59.7% use antiseptics before surgery. Only 54 otolaryngologists, less than half of the subjects in this study (43.5%), always place a tube during the procedure. More physicians who practice in small cities recommend water precautions after MTP when compared to other physicians (p < 0.001). Conclusions The present study reveals that many respondents do not follow some of the recommendations of the current guidelines of the American Academy of

RESUMEN

Introduction Myringotomy and ear tube placement (MTP) is the surgical treatment for otitis media with effusion (OME), and it is the most common surgery performed in children. Several guidelines have been developed to assist in the care of patients who become candidates for MTP. Objectives To evaluate the practice of Brazilian otorhinolarynogologists when performing MTP according to the years of clinical experience. Secondarily, we also want to assess if their practice regarding MTP varied according to the percentage of children treated and the location of their practice. Methods A 30-question survey was sent to otolaryngologists affiliated with the Brazilian Academy of Pediatric Otorhinolaryngology (Academia Brasileira de Otorrinolaringologia Pediátrica, ABOPe, in Portuguese) and/or the Scientific Department of Otorhinolaryngology of the Brazilian Society of Pediatrics (Sociedade Brasileira de Pediatria, SBP, in Portuguese). The questions included were carefully chosen to provide a profile about the practices adopted in the pre-, peri- and postoperative periods of MTP. Results The questionnaire was sent to 208 otolaryngologists, and there were 124 (59.6%) respondents. Of those, 59.7% use antiseptics before surgery. Only 54 otolaryngologists, less than half of the subjects in this study (43.5%), always place a tube during the procedure. More physicians who practice in small cities recommend water precautions after MTP when compared to other physicians ( p < 0.001). Conclusion The present study reveals that many respondents do not follow some of the recommendations of the current guidelines of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) on OME, either perioperatively and postoperatively, or regarding the option of placing a ventilation tube. This part of the care also varied depending on the respondents' work location and experience in the medical practice.

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Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of convergence between signals derived from the mother and the developing fetus/es. Its functions are fuelled by energy generated by mitochondrial oxidative phosphorylation (OXPHOS). The aim of this study was to delineate the role of an altered maternal and/or fetal/intrauterine environment in feto-placental growth and placental mitochondrial energetic capacity. To address this, in mice, we used disruptions of the gene encoding phosphoinositol 3-kinase (PI3K) p110α, a growth and metabolic regulator to perturb the maternal and/or fetal/intrauterine environment and study the impact on wildtype conceptuses. We found that feto-placental growth was modified by a perturbed maternal and intrauterine environment, and effects were most evident for wildtype males compared to females. However, placental mitochondrial complex I+II OXPHOS and total electron transport system (ETS) capacity were similarly reduced for both fetal sexes, yet reserve capacity was additionally decreased in males in response to the maternal and intrauterine perturbations. These were also sex-dependent differences in the placental abundance of mitochondrial-related proteins (e.g., citrate synthase and ETS complexes), and activity of growth/metabolic signalling pathways (AKT and MAPK) with maternal and intrauterine alterations. Our findings thus identify that the mother and the intrauterine environment provided by littermates modulate feto-placental growth, placental bioenergetics, and metabolic signalling in a manner dependent on fetal sex. This may have relevance for understanding the pathways leading to reduced fetal growth, particularly in the context of suboptimal maternal environments and multiple gestation/litter-bearing species.

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Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

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BACKGROUND: Impaired physical function is a common complication in intensive care unit (ICU) patients. However, specific upper limb (UL) function is still poorly studied in this population. OBJECTIVE: To evaluate UL function at discharge and after a 6-month follow-up of individuals hospitalized in the ICU. METHODS: This was a longitudinal prospective 6-month multicentre cohort study with forty-six individuals hospitalized in the ICU undergoing mechanical ventilation for ≥ 48 h (ICU Group) and forty-six healthy individuals matched by sex, age, and socioeconomic status (control Group). The primary outcomes were measurements of UL disability using the Jebsen-Taylor Hand Function Test (JTT) and the Nine Hole Peg Test (NHPT). Secondary outcomes were physical function (Barthel index), muscle strength (Medical Research Council scale and hand grip strength), and quality of life (EuroQol-5 Dimension). All measurements were assessed after ICU discharge and at a 6-month follow-up. RESULTS: The JTT performance time in the ICU group after discharge was worse than that in the control group [121 s (86-165) vs. 54 s (49-61), median (IQR), p<0,001] and was reduced after 6 months [62 s (54-81), p<0,01]. The NHPT performance time at discharge in the ICU group was worse than that in the controls [39 s (33-59) vs. 21 s (20-23), p<0,001] and was reduced after 6 months of follow-up [24 s (21-27), p<0,01]. Physical function, muscle strength and quality of life were reduced after ICU discharge. CONCLUSION: Individuals hospitalized in the ICU presented with reduced UL function at discharge and at the 6-month follow-up.

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Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom (“traditional approach”) and immunization with selected key toxins isolated from the snake venom (“toxin oriented” approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

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A urgência e emergência, por sua vez, se faz como ocorrência imprevista com ou sem risco potencial à vida, onde o indivíduo necessita de assistência e pressupõem atendimento rápido, proporcional a sua gravidade. O presente trabalho tem o objetivo de promover reflexões acerca dos desafios que surgem diante do atendimento a múltiplas vítimas nos serviços médicos de urgência e emergência. Trata-se de uma revisão integrativa da literatura. Realizou-se uma análise de materiais já publicados na literatura e artigos científicos divulgados em bases de dados: Scientific Eletronic Library Online, Medical Literature Analysis and Retrieval System Online e Localizador de informação em Saúde. Foram encontradas nas bases de dados, 25 estudos completos, após a leitura dos resumos, 21 artigos foram selecionados para análise na íntegra, sendo 17 eleitos para integrar a revisão integrativa. Diante dos resultados obtidos, observou que as equipes de atendimento pré- hospitalar vivenciam desafios para atender múltiplas vítimas, e dentro desse paradigma existem várias etapas que devem ser seguidas, que envolvem comunicação desde um protocolo de atendimento inicial ao transporte final. Portanto, observa-se a necessidade de maiores estudos e desenvolvimento de novas tecnologias que auxiliam na assistência a múltiplas vítimas, como também o prepara e atualização dos profissionais.

Urgency and emergency, in turn, is made as an unforeseen occurrence with or without potential risk to life, where the individual needs assistance and quick care, in turn, proportional to its severity. The work of emergency care and the need to respond to problems presented in urgent and emergency services. This is an integrative literature review. An analysis was performed of materials already published in the literature and articles published in databases: Scientific Electronic Library Online, Medical Literature Analysis and Retrieval System Online and Health Information Locator. All studies were complete, after reading the studies,21 articles were selected for full analysis, with 17 studies elected to integrate the integrative review. Results obtained, observed that pre care teams experience challenges to support various hospital communication protocols, and within these paradigms from initial care to transport. Therefore, there is a need for studies and development of technologies that assist in the installation of larger and more up-to-date devices, there is a need for studies and development of new technologies, as well as preparation.

La atención de urgencia y emergencia es un suceso imprevisto con o sin riesgo potencial para la vida, en el que el individuo necesita asistencia y requiere una atención rápida, proporcional a su gravedad. Este documento pretende promover la reflexión sobre los retos que surgen al tratar con múltiples víctimas en los servicios médicos de urgencia y emergencia. Se trata de una revisión bibliográfica integradora. Se ha realizado un análisis de los materiales publicados en la literatura y los artículos científicos divulgados en las bases de datos: Scientific Eletronic Library Online, Medical Literature Analysis and Retrieval System Online y Localizador de información en Salud. Se encontraron en las bases de datos, 25 estudios completos, después de leer los resúmenes, se seleccionaron 21 artículos para el análisis en su totalidad, siendo 17 elegidos para integrar la revisión integradora. A partir de los resultados obtenidos, se observa que los equipos de atención prehospitalaria viven desafíos para atender a múltiples víctimas, y dentro de este paradigma existen varias etapas que deben seguirse, que implican la comunicación desde un protocolo de atención inicial hasta el transporte final. Por lo tanto, se observa la necesidad de realizar más estudios y desarrollar nuevas tecnologías que ayuden en la asistencia a las múltiples víctimas, así como la preparación y actualización de los profesionales.

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Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

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The main protease Mpro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus' pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme's primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel Mpro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that Mpro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of Mpro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.

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O fenômeno de extravasamento de muco, também referido como mucocele, é uma lesão comum da mucosa oral resultante da ruptura de um ducto da glândula salivar com extravasamento da mucina. O principal fator etiológico é o trauma local. A localização mais comum é o lábio inferior, mas outros locais podem ser afetados. Existe a necessidade de diagnóstico diferencial uma vez que, pelo aspecto clínico e localização, as mucoceles assemelham-se a outras lesões que afetam a boca. Diante desse cenário, o objetivo deste trabalho foi relatar dois casos clínicos de fenômeno de extravasamento de muco (mucocele), evidenciando suas características clínicas, histopatológicas, tratamento e prognóstico. Acredita-se que o conhecimento das principais características dessa condição patológica possa auxiliar o clínico na adoção dos principais procedimentos na avaliação, conduta e tratamento desses pacientes.

The mucous retention phenomenon, also called as oral mucocele, is a common lesion of the oral mucosa resulting from the rupture of a salivary gland duct. The main etiological factor is local trauma. The most common location is the lower lip, but other locations can be affected. Differential diagnosis is important because mucoceles resemble other oral lesions. Thus, the aim of the present study was to report two cases of mucocele, demonstrating its clinical and histopathological characteristics, treatment and prognosis.. It is believed that knowledge of the main characteristics of this lesions can help the clinician in carrying out the main procedures in the evaluation, conduct and treatment of these patients.

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Thyroid hormones (THs) are required for the growth and development of the fetus, stimulating anabolism, and oxygen consumption from the early stages of pregnancy to the period of fetal differentiation close to delivery. Maternal changes in the hypothalamic-pituitary-thyroid axis are also well known. In contrast, several open questions remain regarding the relationships between the placenta and the maternal and fetal TH systems. The exact mechanism by which the placenta participates in regulating the TH concentration in the fetus and mother and the role of TH in the placenta are still poorly studied. In this review, we aim to summarize the available data in the area and highlight significant gaps in our understanding of the ontogeny and cell-specific localization of TH transporters, TH receptors, and TH metabolic enzymes in the placenta in both human and rodent models. Significant deficiencies also exist in the knowledge of the contribution of genomic and nongenomic effects of TH on the placenta and finally, how the placenta reacts during pregnancy when the mother has thyroid disease. By addressing these key knowledge gaps, improved pregnancy outcomes and management of women with thyroid alterations may be possible.

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Objetivou-se analisar uma intervenção de cuidado integral em saúde bucal de abordagem comunitária protagonizada por residentes de Odontologia em Saúde da Família inseridos em duas unidades de saúde do campo de Caruaru/PE, segundo o olhar de profissionais, residentes e usuários. A intervenção englobou diagnóstico e levantamento das necessidades odontológicas, ações coletivas em saúde bucal e ações assistenciais para cárie com tratamento restaurador atraumático. Os grupos-alvo formam escolares e moradores de áreas mais remotas nas unidades de Lagoa de Pedra e de Xicuru. O estudo qualitativo utilizou técnica do grupo focal para coleta dos dados. Três grupos foram constituídos: nove profissionais, seis residentes e oito usuários. As entrevistas seguiram um roteiro com perguntas abertas sobre o cuidado e as tecnologias em saúde bucal empregadas no contexto das populações campesinas e acesso/acessibilidade às unidades de saúde. Os dados foram submetidos à análise de conteúdo. Emergiram duas categorias temáticas: dificuldades de acesso aos cuidados em saúde bucal e satisfação com as ações implementadas. Os participantes dos três grupos avaliaram positivamente a iniciativa de estabelecer relação educativa/assistencial participativa abrindo-se a espaços comunitários e valorizaram o enfoque adotado pela intervenção de saúde bucal no enfrentamento dos problemas limitadores do acesso aos serviços odontológicos e às ações coletivas que relataram. Contudo, desinteresse na continuidade das ações realizadas e contrários às práticas comunitárias em saúde bucal foram relatadas no grupo focal dos profissionais. Considera-se persistir fatores profissionais e de estrutura limitadores à garantia dos direitos de acesso à saúde aos usuários de áreas mais remotas às unidades de saúde (AU).

The aimof this study was to analyze a comprehensivecommunity-basedoral health care intervention carried out by residents of Dentistry in Family Health inserted in two health units in the rural area of the municipalityof Caruaru/PE, according to the perspective of professionals, residents and users. The intervention included diagnosis and assessment of dental needs, collective actions in oral health and actions for caries treatment with atraumatic restorative treatment. Target groupswere composed of schoolchildren and users frommore remote areas included in the health units of Lagoa de Pedra and Xicuru. The qualitative study used the focus group technique for data collection. Three groups were formed: nine professionals, six residents and eight users. Interviews followed a script with open questions about oral health care and technologies used in the context of ruralpopulations and access/accessibility to health units. Data were submitted to content analysis. Two thematic categories emerged: difficulties in accessing oral health care and satisfaction with implemented actions. Participants in the three groups positively evaluated the initiative ofestablishinga participatory educational/care relationship by opening up to community spaces and valued the approach adopted by the oral health intervention in dealing with problems that limit access to dental services and the reported collective actions. However, lack of interest in the continuity of actions carried out and contrary to community practices in oral health were reported in the focus group of professionals. There ispersistent presence of professional and structural factors that limit the guarantee of access to health ofusers frommore remote areas included inhealth units.

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Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

RESUMEN

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

RESUMEN

The main protease Mpro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus’ pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme’s primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel Mpro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that Mpro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of Mpro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.