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Background: The COVID-19 pandemic has significantly impacted education systems worldwide, with Brazil being one of the countries with the longest school closures. Over a million children and teenagers have been affected, leading to increased hunger and nutritional deficiencies. This study aimed to implement long-term surveillance of SARS-CoV-2 infections in public and private schools in Campo Grande, Brazil, after returning to in-person classes., Methods: The study involved testing and genomic surveillance at 23 public and private schools in Campo Grande, Mato Grosso do Sul, Brazil, from October 18, 2021 to November 21, 2022. The participants eligible for enrollment were students aged 6-17 years and staff members from school institutions. At the time of collection, participants were asked if they had symptoms in the last two weeks. Whole-genome sequencing of SARS-CoV-2 was conducted to identify circulating variants and to compare them with those detected in the municipality. The demographic data and clinical history of the participants were described, and a logistic regression model was used to understand how the RT-qPCR results could be related to different characteristics., Results: The study included 999 participants, most of whom were women. A total of 85 tests were positive, with an overall positivity rate of 3.2%. The dynamics of case frequency were consistent with those observed in the municipality during the study period. The most common symptoms reported were cough, rhinorrhea, headache, and sore throat. Symptoms were significantly associated with SARS-CoV-2 infection. Eleven lineages were identified in school community samples, with a frequency of occurrence per period similar to that found in the sequences available for the municipality. The most prevalent lineages within the sampling period were BA.2 (59.3%) and BA.5 (29.6%)., Conclusions: Our findings demonstrate that schools can play a crucial role in epidemiological surveillance, helping trigger rapid responses to pathogens such as SARS-CoV-2. Long-term surveillance can be used to track outbreaks and assess the role of children and adults in transmission. It can also contribute to pandemic preparedness, enabling a rapid response to emergencies, such as COVID-19., (© 2024. The Author(s).)

Senna rugosa is a species found in the Cerrado and used in folk medicine as a vermifuge and in the treatment of poisonous snakebites accidents. In this work, we identified the main secondary metabolites present in ethanolic extracts of the leaves (ELSR) and roots (ERSR) of S. rugosa and evaluated the potential cytoprotective effect against cellular macromolecular damage, as well as the cytotoxic properties of the extracts on the K562 and Jurkat leukemic cell lines. The identification of metabolites was carried out by liquid chromatography coupled with mass spectrometry. The antioxidant activities were investigated by direct ABTS •+ and DPPH • radical scavenging methods, protection against oxidative damage in proteins, and DNA. Cytotoxic properties were investigated against healthy cells, isolated from human peripheral blood (PBMC) and leukemic cell lines. The leaf extracts contained catechin, rutin, epigallocatechin derivatives, kaempferol glycosides, luteolin, and dimeric and trimeric procyanidins, while the root extract profile showed obtusichromoneside derivatives, 2-methoxystypandrone, stilbene derivatives, naphthopyranones, and flavanone derivatives. The extracts showed antioxidant activity, with an IC 50 of 4.86 ± 0.51 μg/mL and 8.33 ± 0.90 μg/mL in the ABTS assay for ELSR and ERSR, respectively. Furthermore, in the DPPH • assay, the IC 50 was 19.98 ± 1.96 μg/mL for ELSR and 13.37 ± 1.05 μg/mL for ERSR. The extracts protected macromolecules against oxidative damage at concentrations of 5 μg/mL. The cytotoxicity test against leukemic strains was observed after 24 and 48 h of treatment. After 48 h, results against the K562 cell line demonstrate an IC 50 of 242.54 ± 2.38 μg/mL and 223.00 ± 2.34 μg/mL for ELSR and ERSR, respectively. While against the Jurkat cell line, these extracts showed an IC 50 of 171.45 ± 2.25 μg/mL and 189.30 ± 2.27 μg/mL, respectively. The results pertaining to PBMC viability demonstrated that the extracts showed selectivity for the leukemic cell lines. Together, our results reveal that the leaves and roots of S. rugosa have completely distinct and complex chemical compositions and expand their significant pharmacological potential in oxidative stress and leukemia conditions.

Introduction: With the reopening of schools during the coronavirus disease 2019 (COVID-19) pandemic, it was imperative to understand the role of students and education professionals in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we determined the seroprevalence of the SARS-CoV-2 anti-nucleocapsid antibodies in the school community in Campo Grande, the capital and most populous city of the state of Mato Grosso do Sul (Brazil) and evaluated its association with sex, school level, and school type., Materials and Methods: The survey was carried out in 20 public and private schools in the urban region of Campo Grande using the TR DPP ® COVID-19 immunoglobulin M/immunoglobulin G (IgM/IgG) kit from the Immunobiological Technology Institute (Bio-Manguinhos, Rio de Janeiro, Brazil). Testing was carried out in three periods: from October to December 2021; from March to July 2022; and from August to November 2022. The participants were students aged 6-17 years enrolled in primary or secondary schools and professionals of different ages and roles., Results: During the first testing period, 162 participants were seropositive for the IgM and/or IgG anti-nucleocapsid SARS-CoV-2 antibodies, with an estimated seroprevalence of 19.6% using Bayesian multilevel regression. In the second period, 251 participants were seropositive (estimated seroprevalence, 34.6%), while in the third period, 393 participants were seroconverted (estimated seroprevalence, 56.7%). In 2022, there was an increase in the seroconversion rate compared to that in 2021. The most frequently described acute manifestations in the three periods were fever, headache, sore throat, and runny nose. In terms of the demographic profile, there was no predominance of seropositivity between the sexes, although women represented approximately 70% of the study population. There were also no differences between students and school staff., Discussion: The results made it possible to evaluate the extent of SARS-CoV-2 transmission in the school community through immunity developed against the virus, in addition to providing information about COVID-19 symptoms in children, adolescents, and adults., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stutz, Alcantara, Santos, Torres, Rodrigues, Marcon, Ferreira, Sarti, Oliveira, Mendes, Lemos, Demarchi, Lichs, Zardin, Gonçalves, Guilhermino, Perdomo and Fernandez.)

The Caribbean fruit fly Anastrepha suspensa (Diptera: Tephritidae) is a polyphagous pest causing economic losses in Central America, the Caribbean and South Florida. The parasitoid wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae) is the main parasitoid of A. suspensa in biological control programs. In this study, by modeling with CLIMEX software, climatically suitable areas were projected according to historical climate data. Areas with overlapping optimal climatic suitability for the joint establishment of the pest and parasitoid were mapped, indicating large areas with host presence in North, Central, and South America, with cold stress being the main climatic factor limiting distribution for both species. Tropical regions have the most potential for invasion, with optimal suitability in many areas. Through the projected distributions, this study can target quarantine strategies in areas most susceptible to invasion and establishment of the pest in each country. In addition, classical biological control with the parasitoid in areas with climatic suitability is also recommended., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Background: Toxoplasmosis affects one third of the world population and has the protozoan Toxoplasma gondii as etiological agent. Congenital toxoplasmosis (CT) can cause severe damage to the fetus, including miscarriages, intracranial calcification, hydrocephalus and retinochoroiditis. Severity of CT depends on the gestational period in which infection occurs, and alterations at the cellular level during retinal development have been reported. In this study, we proposed a mouse CT model to investigate the impact of infection on retinal development., Methods: Pregnant females of pigmented C57BL/6 strain mice were infected intragastrically with two T. gondii cysts (ME49 strain) at embryonic day 10 (E10), and the offspring were analyzed at E18., Results: Infected embryos had significantly smaller body sizes and weights than the PBS-treated controls, indicating that embryonic development was affected. In the retina, a significant increase in the number of Ki-67-positive cells (marker of proliferating cells) was found in the apical region of the NBL of infected mice compared to the control. Supporting this, cell cycle proteins Cyclin D3, Cdk6 and pChK2 were significantly altered in infected retinas. Interestingly, the immunohistochemical analysis showed a significant increase in the population of β-III-tubulin-positive cells, one of the earliest markers of neuronal differentiation., Conclusions: Our data suggests that CT affects cell cycle progression in retinal progenitor cells, possibly inducing the arrest of these cells at G2/M phase. Such alterations could influence the differentiation, anticipating/increasing neuronal maturation, and therefore leading to abnormal retinal formation. Our model mimics important events observed in ocular CT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Campos, Magalhães, da Rosa, dos Santos, Fragel-Madeira, Figueiredo, Calaza and Adesse.)

The Caribbean fruit fly, Anastrepha suspensa (Lower, 1862) (Diptera: Tephritidae), is a pest of significant economic importance in Central America and Florida (USA). This study was carried out to examine the influence of climate change on the space-time distribution of A. suspensa on temporal and spatial scales. The CLIMEX software was used to model the current distribution and for climate change. The future distribution was performed using two global climate models (GCMs), CSIRO-Mk3.0 (CS) and MIROC-H (MR), under the emission scenarios (SRES) A2 and A1B for the years 2050, 2080, and 2100. The results indicate a low potential for global distribution of A. suspensa in all scenarios studied. However, tropical areas were identified with high climatic suitability for A. suspensa in South America, Central America, Africa, and Oceania until the end of the century. Projections of areas with climatic suitability for A. suspensa can provide helpful information to develop preventive strategies of phytosanitary management avoiding economic impacts with the introduction of the species., (© 2023. The Author(s) under exclusive licence to International Society of Biometeorology.)

In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 10 4 to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs-TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials.

Despite the advances in in vitro embryo production (IVP) over the years, the technique still has limitations that need to be overcome. In cell cultures, it is already well established that three-dimensional culture techniques are more physiological and similar to the in vivo development. Liquid marble (LM) is a three-dimensional system based on the use of a hydrophobic substance to create in vitro microbioreactors. Thus, we hypothesized that the LM system improves bovine in vitro oocyte maturation and embryo culture. In experiment I, bovine cumulus-oocyte complexes (COCs) were placed for in vitro maturation for 22h in two different groups: control (conventional 2D culture) and LM (three-dimensional culture). We found that oocyte nuclear maturation was not altered by the LM system, however it was observed a decrease in expression of genes important in the oocyte maturation process in cumulus cells of LM group (BCL2, EIF4E, and GAPDH). In experiment II, the COCs were conventionally matured and fertilized, and for culture, they were divided into LM or control groups. There was a decrease in blastocyst rate and cell counting, a down-regulation of miR-615 expression, and an increase in the DNA global methylation and hydroxymethylation in embryos of LM group. Therefore, for the bovine in vitro embryo production, this specific three-dimensional system did not present the advantages that we expected, but demonstrated that the embryos changed their development and epigenetics according to the culture system., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ferronato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The mapping of pastures can serve to increase productivity and reduce deforestation, especially in Amazon Biome regions. Therefore, in this study, we aimed to explore precision agriculture technologies for assessing the spatial variations of soil pH and biomass indicators (i.e., Dry Matter, DM; and Green Matter, GM). An experiment was conducted in an area cultivated with Panicum maximum (Jacq.) cv. Mombaça in a rotational grazing system for dairy buffaloes in the eastern Amazon. Biomass and soil samples were collected in a 10 m × 10 m grid, with a total of 196 georeferenced points. The data were analyzed by semivariogram and then mapped by Kriging interpolation. In addition, a variability analysis was performed, applying both the Normalized Difference Vegetation Index (NDVI) and Normalized Difference Water Index (NDWI) derived from satellite remote sensing data. The Kriging mapping between DM and pH at 0.30 m depth demonstrated the best correlation. The vegetative index mapping showed that the NDVI presented a better performance in pastures with DM production above 5.42 ton/ha -1 . In contrast, DM and GM showed low correlations with the NDWI. The possibility of applying a variable rate within the paddocks was evidenced through geostatistical mapping of soil pH. With this study, we contribute to understanding the necessary premises for utilizing remote sensing data for pasture variable analysis.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicus fur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicus fur protein was able to complement an E. coli fur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus , as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus .

Angiotensin-converting enzyme I (ACE) is a key part of the renin-angiotensin system. Its main function is to regulate blood pressure and the balance of salts in the body. Somatic ACE has two domains, N-C-, each of which has a catalytic site that exhibits 60%sequence identity. The N-domain has a specific action in the hydrolysis of beta-amyloid bodies and angiotensin (1-7), which activates the MAS receptor and triggers anti-thrombotic and anti-inflammatory actions. Our goal was to obtain the catalytic site Ala 361 to Gly 468 of the N domain region, csACE N , without needing purification by chromatography. We employed a method that uses an Elastin-like Polypeptide (ELP) and Intein sequences linked to the peptide of interest. The more differential for obtaining the pure peptide was the cultivation temperatures in the synthesis of ELPcsACE N at 37 °C, with a significant increase in expression. In the purification by ELP precipitation, we recorded the highest efficiency in the concentrations of 0.57 M and 0.8 M of ammonium sulfate buffer. Intein autocleavage study allows removal of the ELP sequence at acidic pH, with the buffers MES and Tris-HCl The present study defined the best conditions for obtaining pure csACE N that the literature has not yet described for peptides. Obtaining pure csACE N aims at future studies for therapeutic use in hypertension, Alzheimer's, and oncology., (Copyright © 2022. Published by Elsevier Inc.)

Propolis is a resinous material rich in flavonoids and involved in several biological activities such as antimicrobial, fungicide, and antiparasitic functions. Conventionally, ethanolic solutions are used to obtain propolis phytochemicals, which restrict their use in some cultures. Given this, we developed an alcohol-free high-performance extractive approach to recover antibacterial and antioxidants phytochemicals from red propolis. Thus, aqueous-solutions of ionic liquids (IL) and eutectic solvents were used and then tested for their total flavonoids, antioxidant, and antimicrobial activities. The surface-responsive technique was applied regarding some variables, namely, the time of extraction, the number of extractions, and cavitation power (W), to optimize the process (in terms of higher yields of flavonoids and better antioxidant activity). After that, four extractions with the same biomass (repetitions) using 1-hexyl-3-methylimidazolium chloride [C 6 mim]Cl, under the operational conditions fixed at 3.3 min and 300 W, were able to recover 394.39 ± 36.30 mg RuE. g -1 of total flavonoids, with total antioxidant capacity evaluated up to 7595.77 ± 5.48 μmol TE. g -1 dried biomass , besides inhibiting the growth of Staphylococcus aureus and Salmonella enteritidis bacteria (inhibition halo of 23.0 ± 1.0 and 15.7 ± 2.1, respectively). Aiming at the development of new technologies, the antimicrobial effect also presented by [C 6 mim]Cl may be appealing, and future studies are required to understand possible synergistic actions with propolis phytochemicals. Thereby, we successfully applied a completely alcohol-free method to obtain antimicrobials phytochemicals and highly antioxidants from red propolis, representing an optimized process to replace the conventional extracts produced until now., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 dos Santos, de Souza Mesquita, Braga and de Rosso.)

Bacterial transcriptome profiling in the presence of plant fluids or extracts during microbial growth may provide relevant information on plant-bacteria interactions. Here, RNA sequencing (RNA-Seq) was used to determine the transcriptomic profile of Herbaspirillum seropedicae strain HRC54 at the early stages of response to sugarcane apoplastic fluid. Differentially expressed gene (DEG) analysis was performed using the DESeq2 and edgeR packages, followed by functional annotation using Blast2GO and gene ontology enrichment analysis using the COG and KEGG databases. After 2 h of sugarcane apoplastic fluid addition to the H. seropedicae HRC54 culture, respectively, 44 and 45 genes were upregulated and downregulated. These genes were enriched in bacterial metabolism (e.g., oxidoreductase and transferase), ABC transporters, motility, secretion systems, and signal transduction. RNA-Seq expression profiles of 12 genes identified in data analyses were verified by RT-qPCR. The results suggested that H. seropedicae HRC54 recognized sugarcane apoplastic fluid as the host signal, and some DEGs were closely involved at the early stages of the establishment of plant-bacteria interactions., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-021-02848-y., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2021.)

Objective: To explore factors associated with social discrimination against users of health services regarding dental aesthetic conditions. Methods: Based on a Brazilian National Survey, multivariate Poisson regressions with robust variance were used to explore the association of outcome discrimination related to different motivations in health services and exposure to sociodemographic and dental variables. Effect modification by complete prosthesis wearing was assessed. Results: Among the 60,200 people interviewed, 11.5% reported being discriminated against in health services. For women, a higher prevalence of discrimination was found among those in the age group of 30-44 years. For both sexes, discrimination was associated with black and brown skin color. Regarding dental characteristics, the higher the tooth loss was, the higher the prevalence of discrimination; however, complete prosthesis wearing presented as a protective factor. Social discrimination was the major motivation for reported discrimination and presented higher prevalence in edentulous individuals who did not wear prosthesis. Conclusion: Dental loss may lead to self-reported discrimination in health care services. The prevalence of discrimination increases when tooth loss increases, and the major reason associated is social discrimination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bulgarelli, dos Santos, Rech, Baumgarten and Goulart.)

Gluconacetobacter diazotrophicus is a species of great agronomic potential due to its growth-promotion traits. Its colonization process in different plants has been reported. However, there have been no studies regarding its structural colonization in elephant grass. This is a fast-growing C4-Poaceae plant, and its application in Brazil is mainly aimed at feeding dairy cattle, due to its high nutritional value. Also, in the last decade, this grass has been applied in the production of biofuels. The present study aimed to monitor the colonization process of strain LP343 of G. diazotrophicus inoculated in elephant grass seedlings of PCEA genotype, by using a mCherry-tagged bacterium. Samples of roots and shoots collected at different periods were visualized by confocal laser-scanning microscopy. The colony-counting assay was used to compare the number of cells recovered in different niches and a qPCR was performed for the quantification of endophytic cells in root and shoot tissues. Results suggested that the strain LP343 quickly recognized the PCEA roots as host, attached to the elephant grass roots at 6 h, and 7 days after inoculation were able to colonize the xylem vessels of roots and shoots of elephant grass. This study advances our knowledge about the colonization process of G. diazotrophicus species in elephant grass, contributing to future studies involving the plant-bacteria interaction cultivated under gnotobiotic conditions., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Demands as to productivity transform the work environment more and more stressful, and negatively impact on the motivation of workers and the performance of their work activities. Initiatives seek to consolidate advances in policies for comprehensive care for workers health, with actions to prevent injuries and promote health. Offering workers a Labor Gymnastics Program (PGL), which is not restricted to attention to musculoskeletal disorders, becomes a tool to encourage change in habits., Aim: To identify possible changes in workers habits after the implementation of a PGL., Method: 41 workers in the administrative area and support from a teaching hospital in the municipality of Santa Cruz do Sul answered the questionnaire. The questionnaire includes variables on eating habits, displacement and physical activity, applied before and after intervention. The sessions were held during the workday, with frequency of three times a week, on alternate days, for 4 months, composed of physical activities and information on dietary reeducation, postural education, body awareness and the importance of regular physical activity., Results: There is a predominance of females (61.0%), and age of 33.5±10.2 years. Changes in some habits were identified: increase in the number of workers who practice regular physical activity (p=0.01), increase in the duration and frequency of activity (p=0.04), and maintenance / reduction in the frequency of consumption of "Embedded" (p=0.03)., Conclusion: It was possible to identify positive changes in workers habits after the implementation of the PGL, suggesting that it is an important strategy in the field of health promotion.

Background: The state of health of rural workers is influenced by the living conditions to which they are subjected, including social, economic, technological and organizational aspects. Given the scarcity of studies on this population of workers, establishing their profile is necessary., Objectives: To analyze cardiovascular risk according to demographic factors and anthropometric status of rural workers under the Pardo River Valley Regional Development Council (COREDE-VRP)., Methods: Cross-sectional analytical study with rural workers in five municipalities in the COREDE-VRP southern region. We administered a structured questionnaire for lifestyle socioeconomic information, physical activity and self-reported health. Anthropometric measurements, resting heart rate and electrocardiogram (ECG) were performed to analyze heart rate variability (HRV)., Results: Women exhibited higher cardiovascular risk, which in turn did not differ as a function of age, marital status, socioeconomic status or lifestyle. We found a relationship between cardiovascular risk and anthropometric measurements, but not with cardiovascular variables., Conclusion: Women exhibited higher cardiovascular risk, which was not associated with marital status, socioeconomic status, alcohol use, smoking, sleep disorders or physical activity. Therefore, we emphasize the relationship between cardiovascular risk and anthropometric variables, as well as the lack of association with heart rate and autonomic imbalance.

Different sampling strategies, analytic alternatives, and estimators have been proposed to better assess the characteristics of different hard-to-reach populations and their respective infection rates (as well as their sociodemographic characteristics, associated harms, and needs) in the context of studies based on respondent-driven sampling (RDS). Despite several methodological advances and hundreds of empirical studies implemented worldwide, some inchoate findings and methodological challenges remain. The in-depth assessment of the local structure of networks and the performance of the available estimators are particularly relevant when the target populations are sparse and highly stigmatized. In such populations, bottlenecks as well as other sources of biases (for instance, due to homophily and/or too sparse or fragmented groups of individuals) may be frequent, affecting the estimates.In the present study, data were derived from a cross-sectional, multicity RDS study, carried out in 12 Brazilian cities with transgender women (TGW). Overall, infection rates for HIV and syphilis were very high, with some variation between different cities. Notwithstanding, findings are of great concern, considering the fact that female TGW are not only very hard-to-reach but also face deeply-entrenched prejudice and have been out of the reach of most therapeutic and preventive programs and projects.We cross-compared findings adjusted using 2 estimators (the classic estimator usually known as estimator II, originally proposed by Volz and Heckathorn) and a brand new strategy to adjust data generated by RDS, partially based on Bayesian statistics, called for the sake of this paper, the RDS-B estimator. Adjusted prevalence was cross-compared with estimates generated by non-weighted analyses, using what has been called by us a naïve estimator or rough estimates.

Stingless bees produce geopropolis, which is popularly described for its medicinal properties, but for which few scientific studies have demonstrated pharmacological effects. The objective of this study was to investigate the chemical composition of the geopropolis of Melipona quadrifasciata anthidioides and to evaluate its antioxidant, antimutagenic, anti-inflammatory, and antimicrobial activities. The composition of the hydroethanolic extract of geopropolis (HEG) included di- and trigalloyl and phenylpropanyl heteroside derivatives, flavanones, diterpenes, and triterpenes. HEG showed antioxidant action via the direct capture of free radicals and by inhibiting the levels of oxidative hemolysis and malondialdehyde in human erythrocytes under oxidative stress. HEG also reduced the frequency of gene conversion and the number of mutant colonies of S. cerevisiae . The anti-inflammatory action of HEG was demonstrated by the inhibition of hyaluronidase enzyme activity. In addition, HEG induced cell death in all evaluated gram-positive bacteria, gram-negative bacteria, and yeasts, including clinical isolates with antimicrobial drug resistance. Collectively, these results demonstrate the potential of M. q. anthidioides geopropolis for the prevention and treatment of various diseases related to oxidative stress, mutagenesis, inflammatory processes, and microbial infections.

Objective: To describe the safety and efficacy of pegvisomant therapy and the predictors of treatment response in acromegaly patients at a single tertiary reference center in Brazil., Materials and Methods: We retrospectively reviewed the clinical, hormonal and radiological data of acromegaly patients treated with pegvisomant in our center. We also evaluated the presence of the d3 isoform of the growth hormone receptor (d3GHR)., Results: Twenty-seven patients were included (17 women). Pegvisomant was used in combination with octreotide LAR in 20 patients (74%), in combination with cabergoline in one (4%) and as monotherapy in six (22%). IGF-I normalization was achieved in 23 patients (85%). Mild and transitory elevation of liver enzymes was observed in two patients (7.4%), tumor growth in one (3.4%) and lipodystrophy in two (7.4%). One patient stopped the drug due to headaches. The GHR isoforms were evaluated in 14 patients, and the presence of at least one d3GHR allele was observed in 43% of them, but it was not a predictor of treatment response. Only pre-treatment IGF-I level was a predictor of treatment response., Conclusion: Pegvisomant treatment was highly effective and safe in our series of Brazilian patients. A better chance of disease control can be expected in those with lower pre-pegvisomant IGF-I levels.

Background: Shortened treatment regimens for tuberculosis are under development to improve treatment outcomes and reduce costs. We estimated potential savings from a societal perspective in Brazil following the introduction of a hypothetical four-month regimen for tuberculosis treatment., Methods: Data were gathered in ten randomly selected health facilities in Rio de Janeiro. Health service costs were estimated using an ingredient approach. Patient costs were estimated from a questionnaire administered to 126 patients. Costs per visits and per case treated were analysed according to the type of therapy: self-administered treatment (SAT), community- and facility-directly observed treatment (community-DOT, facility-DOT)., Results: During the last 2 months of treatment, the largest savings could be expected for community-DOT; on average USD 17,351-18,203 and USD 43,660-45,856 (bottom-up and top-down estimates) per clinic. Savings to patients could also be expected as the median (interquartile range) patient-related costs during the two last months were USD 108 (13-291), USD 93 (36-239) and USD 11 (7-126), respectively for SAT, facility-DOT and community-DOT., Conclusion: Introducing a four-month regimen may result in significant cost savings for both the health service and patients, especially the poorest. In particular, a community-DOT strategy, including treatment at home, could maximise health services savings while limiting patient costs. Our cost estimates are likely to be conservative because a 4-month regimen could hypothetically increase the proportion of patients cured by reducing the number of patients defaulting and we did not include the possible cost benefits from the subsequent prevention of costs due to downstream transmission averted and rapid clinical improvement with less side effects in the last two months.

Introduction: Brazilian spotted fever is an emerging zoonosis notified mainly in the Southeast of Brazil, especially due to its high level of lethality., Objective: To analyze the epidemiological and spatial pattern of the disease in the municipality of Valinhos (106,793 inhabitants), São Paulo, Southeastern region of Brazil, in the period between 2001 and 2012., Methods: All laboratory-confirmed cases with likely site of infection in the city (n = 49) notified in the Brazilian Case Registry Database were studied. Sites were geocoded using the cartographic base of the city and Google Earth (geographic coordinates) with correction according to the Brazilian Geodetic System. We used the Kernel estimator to analyze the density of the cases on the map. Land cover and distance to basins of all cases were analyzed. Information about tick species and primary hosts were obtained from reports of the Superintendence of Control of Endemic Diseases., Results: Seasonality of the disease was observed with the highest incidence from June to November, and in 2005 and 2011. The most affected groups were men (79.6%) aged 20-49 years old (49%). Lethality was found to be 42.9%. Maps showed the progressive registration of cases in the urban area. Capybaras were reported as the main primary host, and Amblyomma cajennense was identified in probable sites of infection during field investigation. The likely sites of infection were mostly located near basins, dirty pastures, and bordering woods., Conclusions: The transmission pattern of Brazilian spotted fever in Valinhos is similar to that in other cities in the region, where capybara is the main primary host and an amplifier of R. rickettsii. Over the years, a higher occurrence of cases has been identified in the urban area of the city.

Obesity associated with a sedentary lifestyle can lead to changes in the immune system balance resulting in the development of inflammatory diseases. The aim of this study was to compare lymphocyte activation mechanisms between overweight children practicing regular circus physical exercises with non-exercised children. The study comprised 60 pubescent children randomly divided into 4 groups: Overweight Children (OWC) (10.67 ± 0.22 years old), Overweight Exercised Children (OWE) (10.00 ± 0.41 years old), Eutrophic Children (EC) (11.00 ± 0.29 years old) and Eutrophic Exercised Children (EE) (10.60 ± 0.29 years old). OWE and EE groups practiced circus activities twice a week, for 4.3 ± 0.5 and 4.4 ± 0.5 months, respectively. Percentage of T regulatory cells (Treg) and the expression of CD95 and CD25 in CD4+ lymphocytes were evaluated by flow cytometry. Lymphocyte proliferation capacity was measured by [14C]-thymidine incorporation and mRNA expression of IL-35, TGF-beta, IL-2 and IL-10 by real-time PCR. Lymphocyte proliferation was higher in OWC and OWE groups compared with the EC (3509 ± 887; 2694 ± 560, and 1768 ± 208 cpm, respectively) and EE (2313 ± 111 cpm) groups. CD95 expression on lymphocytes was augmented in the EC (953.9 ± 101.2) and EE groups (736.7 ± 194.6) compared with the OWC (522.1 ± 125.2) and OWE groups (551.6 ± 144.5). CTLA-4 expression was also lower in the OWC and OWE groups compared with the EC and EE groups. Percentage of Treg, IL-35, and IL-10 mRNA expression were lower in the OWC and OWE groups compared with the EC and EE groups. In conclusion, overweight children present altered immune system balance characterized by elevated lymphocyte proliferation due to a decrease in T regulatory cell percentage. These effects were partially reverted by moderate physical exercise, as demonstrated by decreased lymphocyte proliferation.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu., (Copyright © 2015 Leung et al.)

Context: No long-term studies have compared centrally acting drugs for treating obesity., Objective: To compare the efficacy and safety of diethylpropion (DEP), fenproporex (FEN), mazindol (MZD), fluoxetine (FXT) and sibutramine (SIB) in promoting weight loss., Design and Setting: A prospective, randomized, placebo (PCB)-controlled study conducted at a single academic institution., Patients: A total of 174 obese premenopausal women., Intervention: Participants randomly received DEP 75 mg (n=28), FEN 25 mg (n=29), MZD 2 mg (n=29), SIB 15 mg (n=30), FXT 20 mg (n=29) or PCB (n=29) daily over 52 weeks. Diet and physical activity were encouraged., Main Outcome Measures: The primary endpoints were changes in body weight and the proportion of women who achieved at least 5% weight loss by week 52 in the intent-to-treat population. Other measurements included anthropometry, safety, metabolic and cardiovascular parameters., Results: Weight loss was greater than PCB (-3.1±4.3 kg) with DEP (-10.0±6.4 kg; P<0.001), SIB (-9.5±5.9 kg; P<0.001), FEN (-7.8±6.9 kg; P<0.01) and MZD (-7.4±4.9 kg; P<0.01) but not with FXT (-2.5±4.1 kg). Ten (33.3%) women lost⩾5% of their initial weight with PCB, compared with 20 (71.4%; P<0.001) with DEP, 20 (69%; P<0.02) with FEN, 21 (72.4%; P<0.01) with MZD, 22 (73.3%; P<0.001) with SIB and 10 (35.5%) with FXT. Each medically treated group experienced more adverse events compared with PCB (P<0.001). Compared with PCB, constipation was more prevalent with DEP, SIB and MZD (P<0.01); anxiety was more prevalent with DEP (P=0.01); and irritability occurred more frequently with DEP and FEN (P=0.02). Significant improvements in the depression and anxiety scores, binge-eating episodes and quality of life correlated with weight loss., Conclusion: The centrally acting drugs DEP, FEN, MZD and SIB were more effective than PCB in promoting weight loss in obese premenopausal women, with a satisfactory benefit-risk profile.

Background: Esophageal involvement by Chagas' disease causes a significative decrease in the number of neurons of the esophageal myenteric plexus, causing an impairment of esophageal motility with the same alterations described in primary esophageal achalasia. There is also a longer duration of pharyngeal transit, which might be consequent of the involvement of the central control of swallowing by the disease, or an adaptation of the pharynx to the difficult bolus transit through the esophagus, which could contribute to the complaint of dysphagia., Methods: We studied, by videofluoroscopy, the sequence and timings of pharyngeal bolus transit in 16 patients with esophageal involvement by Chagas' disease and 12 healthy volunteers. Each subject swallowed in duplicate 5 mL and 10 mL of liquid and paste boluses., Results: There was no difference between Chagas' disease patients and normal volunteers in the sequence and timing of events associated with pharyngeal bolus flow, for liquid and paste boluses, and for 5 mL and 10 mL., Conclusion: The timing and sequence of swallow pharyngeal events of patients with Chagas' disease do not differ from that of control subjects, which suggested that the central control of swallowing is not impaired by the disease.

Primary cutaneous lymphomas have different clinical behavior and prognosis than systemic lymphomas of similar histological subtype. About 30% of non-Hodgkin lymphomas involve extranodal tissues, the skin being the second most affected body organ after the gastrointestinal tract (approximately 18%). According to the EORTC (European Organization for Research and Treatment of Cancer) classification, the lymphoma centrofollicular is indolent, since the lesions increase in size slowly over the years and spread to extracutaneous sites is uncommon. Based on this, radiotherapy has been considered the treatment of choice by many studies. We present the case of a patient who had cutaneous centrofollicular scalp lymphoma for 1 year and showed good response to radiotherapy.

Background: Chagas' disease causes dysphagia, regurgitation and retention of food in the esophageal body. Patients have longer pharyngeal clearance, which might be consequent of the involvement of the central nervous system or an adaptation to the esophageal transit impairment. If there is central nervous system involvement by the disease, we expect a larger difference in the oral and pharyngeal phases of swallowing between two consecutive swallows than that seen in controls. Our objective was to evaluate the difference of oral and pharyngeal transit duration between two consecutive swallows in patients with Chagas' disease compared with controls., Methods: By videofluoroscopy, the duration of oral and pharyngeal transit, pharyngeal clearance, upper esophageal sphincter transit, hyoid movement and oropharyngeal transit was measured in 17 patients with Chagas' disease and 15 asymptomatic volunteers. Each subject swallowed in duplicate and in sequence 5 mL and 10 mL of barium liquid and 5 mL and 10 mL of barium paste boluses. The differences were calculated between the two swallows of each volume and consistency in patients and controls., Results: There were no differences between controls and patients in the values of the differences between the two consecutive swallows, except for the hyoid movement duration of the 5 mL liquid bolus, causing a higher difference in controls than in patients., Conclusion: Oral and pharyngeal transit variation between two consecutive swallows is similar between patients with Chagas' disease and controls, which suggests that the longer pharyngeal clearance duration previously described is not a consequence of impairment of the central nervous system control of swallowing.

The aim of this study was to determine the prevalence of human papillomavirus (HPV) in a women population living within the state of Amazonas, Brazil, and to determine the viral genotypes found. The study included 361 sexually active women over 18 years of age. We performed the Pap test and the molecular diagnosis for HPV DNA using polymerase chain reaction (PCR). The amplicons obtained were sequenced in automatic sequencer for genotyping. The presence of HPV DNA was found in 29.1% (105) of the women. Only 321 women presented satisfactory slides for cytological diagnosis, 97.9% (314) had normal cytology (negative for cancer), and 2.1% (7) had abnormal cytology (4 ASCUS, 1 LSIL, and 2 HSIL). The types more frequently found were HPV 16 (58.1%) and HPV 58 (20.0%). Additionally, we found more 13 types of HPV. Compared with previous studies in Brazil, our data confirmed a high prevalence and genotypic diversity of HPV in Brazilian women.

Objective: to evaluate the average resulting area from the Pressure x Time curves resulting from the manometric anorectal exam and compare it with the support capacity of voluntary squeeze., Materials and Methods: the data set was represented by data from 11 exams from continent patients and eight exams from patients with grade III fecal incontinence. The manometric curves were delineated, and the areas and support capacity of voluntary squeeze calculated, by means of the R computer language and the algorithm developed., Results: the resulting averages for support capacity of voluntary squeeze in continent patients and patients with grade III fecal incontinence were 33.07 seconds and 30.76 seconds (p>0.05) and the averages for area were 2362.04 mmHg x second and 947.92 mmHg x second (p<0.05), respectively., Conclusion: the average resulting area is able to differentiate continent patients from incontinent and is shown to be a possible parameter in the analysis of biomechanical behavior related to the mechanisms of anorectal continence.

Background: This study investigated the performance of patients with idiopathic Parkinson's disease (PD) without dementia for incidental recognition memory and the effect of encoding strategies on contextual memory., Methods: The authors studied 21 patients with PD (ages 60-85, 12 women; Hoehn and Yahr I-III, Activities of Daily Living 70%-100%) and 22 healthy controls (ages 60-84, 18 women). Participants completed the vocabulary subtest of the Wechsler Adult Intelligence Scale and the Wisconsin Card Sorting Test (WCST). To assess the incidental recognition memory for item (object) and context (location of the object), participants of each group were assigned to 1 of 2 encoding conditions: ( a) an incidental associative instruction to bind the object to its location or (b) a nonassociative, nonspecific instruction., Results: PD patients showed performance comparable to the control group's on the vocabulary subtest and WCST. In contrast to controls, PD patients were unable to take advantage of the associative encoding instruction, which also had a deleterious effect on item recognition., Conclusion: This sample of participants with PD showed diminished item and context recognition memory and an impaired ability to use incidental memory encoding strategy, suggesting a compromised cognitive reserve. The fact that these alterations occurred in early stages of PD, and prior to more general cognitive alterations such as executive dysfunction, should be considered in the management of patients by using specific cognitive rehabilitation interventions.

Bovine herpesvirus type 5 (BoHV-5) is the agent of meningoencephalitis, an important disease of cattle in South America. The neuropathogenesis of BoHV-5 infection is poorly understood and most previous research focused on the role of envelope glicoproteins in neurovirulence. Thymidine kinase (TK) is a viral enzyme necessary for virus replication in neurons and, therefore, represents a potential target for virus attenuation. The selection and characterization of BoHV-5 variants resistant to the nucleoside analog brivudin (BVDU), which selects TK-defective viruses is here described. Several BVDU-resistant clones were obtained after multiple passages in tissue culture in the presence of BVDU and one clone (BoHV-5/R-27) was further characterized. The selected clone replicated to similar titers and produced plaques with similar size and morphology to those of wild-type virus (SV507/99). The genetic stability of the resistant virus was demonstrated after ten passages in cell culture in the absence of the drug. Moreover, the drug-resistant virus showed reduced virulence in a rabbit model: virus inoculation in four rabbits did not result in disease, in contrast with 75% morbidity (3/4) and 50% mortality (2/2) among rabbits inoculated with the parental virus. These results demonstrate that BoHV-5 is sensitive to BVDU and that drug-resistant mutants can be readily selected upon BVDU treatment. BVDU-resistant mutants, likely defective in TK, retained their ability to replicate in tissue culture yet were attenuated for rabbits. This strategy to obtain TK-defective BoHV-5 may be useful to study the role of TK in BoHV-5 neuropathogenesis and for vaccine development.

The design, synthesis, and X-ray crystallographic analysis of three simple diaryl-urea based anion receptors possessing an amide moiety on one of the aryl groups, and an electron withdrawing CF(3) group on the other, is described. The three receptors differ only in the position of the amide functionality relative to the hydrogen bonding urea moiety (being para, meta, and ortho for 1, 2, and 3, respectively). This simple modification was shown to have a significant effect on the anion recognition ability, the strength of the recognition process, and the stoichiometry (host/guest) for these sensors. We demonstrate, by using both UV-vis absorption and (1)H NMR spectroscopy, that these factors are caused by the ability of the amide moiety to both modulate the anion binding selectivity and the sensitivity of the urea moiety. We also demonstrate that, in the case of 1 and 2, this anion recognition at the urea moiety leads to concomitant activation (through enhanced inductive effect) in the amide functionality toward anions, which leads to the formation of an overall 1:2 (sensor/anion) binding stoichiometry for these receptors. This "activation" we describe as being an example of a "positive allosteric activation" by the urea site, caused directly by the first anion binding interaction, which to the best of our knowledge, has not been previously demonstrated for anion recognition and sensing.

The aim of this research was to verify the incidence of endocrine dysfunction associated with mercury intoxication in the hypothalamus-pituitary reproductive system of normally cycling or castrated female rats and the possible protective action of estrogen replacement therapy. We found no differences in the frequency of estrus cycle stages (diestrus I, diestrus II, proestrus, and estrus) in normally cycling female rats during 54 days of daily oral administration of 0.004, 0.02, and 1 mg/kg MeHgCl. Conversely, the higher dose (1 mg/kg) induced a significant decrease in content of luteinizing hormone releasing hormone (LHRH) into the medial hypothalamus when administered daily during 3 days in ovariectomized rats. This effect was associated with increased levels of mercury found in the anterior pituitary gland and medial hypothalamus, rather than the anterior and posterior hypothalamus, striatum or cerebellum. A decrease in plasma levels of luteinizing hormone (LH) was also detected after administration of 7.5 mg/kg MeHgCl. These disturbances in LHRH and LH secretion induced by mercury were abolished or superimposed (respectively) by estrogenic replacement therapy (0.025 mg/kg 17beta estradiol cypionate, intramuscular). These effects were associated with a significant reduction in mercury content of the anterior pituitary gland and medial hypothalamus, suggesting a protective estrogenic effect.

Background and Objectives: Guillain-Barré syndrome following measles vaccination is uncommon. Diagnosis is often delayed, leading to increased morbidity. This report describes an advanced Guillain-Barré case and the special approaches required during anesthesia., Case Report: Male patient, four years old, with Guillain-Barré syndrome diagnosed at 1 year of age, submitted to gastrostomy under uneventful general anesthesia with sevoflurane, without neuromuscular blockers., Conclusions: The case highlights the low frequency with which this syndrome so important for anesthetic practice is diagnosed, post-vaccination adverse events, the best choice for the anesthetic team and complications of pediatric Guillain-Barré syndrome.

In the coarctation hypertension model, we have shown that chronic treatment with losartan causes both normalization of impaired reflex control of heart rate and partial correction of the depressed aortic nerve activity/pressure relationship, even with the persistence of hypertension. In the present study, we analyzed the effects of angiotensin II blockade on the efferent pathways of coarcted and sham-operated groups treated chronically with vehicle or losartan (10 mg/kg per day PO). Hypertension was induced by subdiaphragmatic aortic coarctation, and the treatments lasted 9 days (4 control and 5 experimental days). On day 5, autoregressive power spectral analysis was performed on heart rate recordings made in conscious rats. Other groups were used for sympathetic splanchnic nerve activity recordings made simultaneously with pressure (anesthetized rats) at basal condition and during loading/unloading of baroreceptors. Losartan treatment induced a significant reduction in basal pressure but did not interfere with the development of hypertension (similar pressure increases of 24% and 28% over control values in losartan and vehicle groups, respectively). In vehicle-treated rats, establishment of hypertension was accompanied by a marked change in power spectral density from high- (1.19+/-0.06 Hz, 33+/-6%) to low-frequency components (0,42+/-0.03 Hz, 54+/-6%), with increased low-frequency-to-high-frequency ratio. When compared with sham-operated vehicle-treated rats, there was also increase in the gain of sympathetic activity/pressure relationship, with displacement of lower plateau toward high levels of sympathetic activity. No changes in the power spectral density and sympathetic activity/pressure relationship were observed when hypertension developed in the presence of chronic angiotensin type 1 (AT(1)) receptor blockade. The data suggest that angiotensin II, activated during the establishment of coarctation hypertension, acts via AT(1) receptors to alter sympathovagal balance, facilitating the sympathetic outflow to heart and peripheral circulation during baroreceptors unloading. Data also indicate that the observed effects are not conditioned by preexisting pressure levels.

The ubiquitin-proteasome system is required in growth hormone receptor (GHR) endocytosis. For cytokine receptors, which lack intrinsic tyrosine kinase activity, signal transduction is initiated by the activation of a member of the Janus kinase (JAK) family. Previously, we have shown that GHR and JAK2 tyrosine (de) phosphorylation are regulated via the ubiquitin system. In this study, we examined the role of JAK2-mediated signal transduction in GHR internalization and down-regulation. Mutation of the attachment site for JAK2, box-1, in the GHR cytoplasmic tail resulted in the complete absence of GHR and JAK2 phosphorylation. This modification did not alter the rate and extent of receptor-bound growth hormone internalization as compared with a functional GHR, nor did it change its turnover and transport to the plasma membrane. In addition, the receptor was still normally ubiquitinated and remained dependent on both an intact ubiquitin system and proteasomal action for its internalization. Thus, GHR ubiquitination, endocytosis, and degradation occur independently of GHR signal transduction via JAK2. We conclude that whereas endocytosis and degradation require the ubiquitin system, they are independent of GHR signal transduction.

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.

The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHR-truncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH.GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.

The ubiquitin conjugation system is involved in ligand-induced endocytosis of the growth hormone receptor (GHR) via a cytosolic 10-amino acid ubiquitin-dependent endocytosis motif. Herein, we demonstrate that the proteasome is also involved in growth hormone receptor down-regulation. Ligand-induced degradation was blocked in the presence of specific proteasomal inhibitors. In addition, growth hormone (GH) internalization was inhibited, whereas the transferrin receptor cycle remained unaffected. A truncated GHR entered the cells independent of proteasome action. In addition, we show that GH internalization is independent of the presence of lysine residues in the cytosolic domain of the receptor, whereas its internalization can still be inhibited by proteasomal inhibitors. Thus, GHR internalization requires proteasome action in addition to an active ubiquitin conjugation system, but ubiquitination of the GHR itself seems not to be required.

In the chronic phase of coarctation hypertension (CH) we have shown both reduction in baroreceptor sensitivity (Hypertension. 1992;19[suppl II]:II-198-II-201.) and normalization of the depressed baroreceptor reflex control of heart rate, even with the persistence of hypertension in losartan-treated animals (Am J Physiol. 1995;269:H812-H818). In the present study we analyzed the effects of angiotensin II blockade on afferent aortic nerve activity of CH and sham-operated groups treated chronically with vehicle or losartan (10 mg/kg per day p.o.). CH was induced by subdiaphragmatic aortic coarctation, and the treatments lasted 8 days (4 control and 4 experimental days). Aortic pressure (conscious rats) and aortic nerve activity simultaneous to pressure (anesthetized rats) were recorded on the fourth day of the experimental period. Losartan-treated rats showed reduced tail pressure (104+/-3 versus 117+/-3 mm Hg in the vehicle group). In both groups, aortic coarctation caused a significant increase in pressure (25% and 28%, respectively) and a depression of the aortic nerve activity/pressure relationship when compared with sham-operated coarcted animals. In the physiological range of pressure changes, the depression was significantly smaller after losartan treatment (3.30+/-0.33 versus 2.18+/-0.37%/mm Hg in the losartan- and vehicle-treated CH groups, respectively, versus 5.05+/-0.33%/mm Hg in the sham-operated vehicle-treated group). Angiotensin type 1 (AT1) receptor blockade was also accompanied by reduced variability of the afferent discharge. The data suggested that apart from its pressure effect, angiotensin II acts at AT1 receptors to decrease the sensitivity of aortic afferents during physiological (+/-10 mm Hg) increases and decreases in pressure. Thus, angiotensin II may contribute to reductions of baroreceptor gain in chronic hypertension.