Análise dos mecanismos reguladores dos processos de polarização e germinação de esporos e, desenvolvimento de gametófitos jovens de Gelidium floridanum sob efeito da radiação ultravioleta e do metal pesado cádmio

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2014.Gelidium floridanum W.R. Taylor é uma alga vermelha de grande importância comercial por ser fonte de ágar com melhor qualidade. Este estudo teve como objetivo ampliar os conhecimentos sobre a germinação de tetrásporos e os efeitos da radiação ultravioleta e do metal pesado cádmio (Cd) nos gametófitos jovens de G. floridanum. Para a análise dos mecanismos reguladores da polarização e germinação, os tetrásporos foram cultivados com brefeldina A (BFA), colchicina e citocalasina, sendo analisados por microscopia de luz (ML), fluorescência e eletrônica de trasmissão (MET). Com a utilização da BFA, a germinação dos esporos não ocorreu, a BFA levou a uma desmontagem das cisternas do Golgi com formação de regiões vesiculares no citoplasma, bloqueando a secreção do Golgi para a formação do tubo germinativo e deposição da parede celular. O tubo germinativo, nas amostras controle, é formado pela incorporação de vesículas derivadas do Golgi, a secreção das vesículas e organização dos corpos de Golgi são processos básicos e essenciais na adesão e formação do tubo. A BFA bloqueou a secreção de proteínas e de polissacarídeos amorfos da matriz, impedindo a germinação dos tetrásporos. Com os inibidores do citoesqueleto, citocalasina e colchicina, a germinação de tetrásporos de G. floridanum também foi afetada. Estes agentes causaram diminuição das taxas de germinação além da malformação dos tubos germinativos e no caso da citocalasina alteração no formato dos cloroplastos. Pode-se sugerir que tanto os microtúbulos quanto os filamentos de actina são reguladores dos processos de polarização e germinação de tetrásporos de G. floridanum. Para os efeitos da radiação ultravioleta, gametófitos jovens de G. floridanum foram cultivados em laboratório e expostos a radiação fotossinteticamente ativa (PAR) de 80µmol fótons m-2 s-1 e PAR+UVA+UVB durante 3 h por dia, por um período de 3 dias. As amostras foram processadas para ML e MET para análise ultraestrutural, bem como análise das taxas de crescimento, teor de pigmentos fotossintéticos, identificação dos carotenóides e avaliação do desempenho fotossintético. PAR + UVA + UVB promoveu aumento da espessura da parede celular, acúmulo de grãos de amido das florídeas no citoplasma e rompimento da organização interna do cloroplasto. As amostras expostas a PAR + UVA + UVB mostraram uma redução na taxa de crescimento de 97%, aumento dos carotenóides, diminuição dos pigmentos fotossintetizantes, em particular, a ficoeritrina e aviiialoficocianina, e consequente diminuição do desempenho fotossintético observados pela redução da taxa de tranporte de elétrons (ETR) e ETRmax. Para os efeitos do Cd, gametófitos jovens foram cultivados durante 7 dias com concentrações de 7,5 e 15 µM do Cd. Após o período experimental, foram realizadas análises de ML, confocal e eletrônica de varredura (MEV), taxas de crescimento, quantificação dos pigmentos fotossintetizantes, carotenóides e a fluorescência da clorofila a. As amostras tratadas apresentaram diminuição das taxas de crescimento, com despigmentação dos talos, redução dos pigmentos fotossintetizantes, resultando num descréscimo da taxa de transporte de elétrons e da autofluorescência dos cloroplastos. Além disso, o Cd aumentou a quantidade de grãos de amido das florídeas e afetou a distribuição do conteúdo proteico no citoplasma. O metal cádmio é altamente tóxico, causando fotoinibição crônica para a espécie. Pode-se concluir que tanto os corpos de Golgi como o citoesqueleto, filamentos de actina e microtúbulos, estão envolvidos nos processos de polarização e formação do tubo germinativo durante a germinaçãos de tetrásporos e, que gametófitos jovens de G. floridanum quando expostos tanto a radiação quanto ao Cd apresentam alterações no metabolismo e na estrutura celular que afetam seu desenvolvimento.Abstract : Gelidium floridanum W. R. Taylor is a red alga of great commercial importance as a source of high-quality agar. This study aimed to examine the germination of tetraspores and the effects of ultraviolet radiation (UVR) and heavy metal cadmium (Cd) on young gametophytes of G. floridanum. To analyze the regulatory mechanisms of polarization and germination, the tetraspores were cultured with brefeldin A (BFA), colchicine and cytochalasin, followed by analysis under light (LM), fluorescence and transmission electron microscopy (TEM). The use of BFA blocked the secretion of protein and amorphous matrix polysaccharides. It also blocked secretion from Golgi bodies, affecting germ-tube formation, as well as, cell wall deposition, and leading to the disassembly of the Golgi cisternae with regions of vesicular formation in the cytoplasm. These events effectively prevented tetraspore germination. Tetraspore germination was also affected by the use of cytochalasin and colchicine, which are inhibitors of cytoskeleton. These agents caused a decrease in germination rates and, in high concentrations, complete inhibition of germination. In addition, the germ tube becames malformed and, in the case of cytochalasin, the shape of chloroplasts was altered. It can be suggested that both microtubule and actin are regulators of polarization and germination in tetraspores of G. floridanum. Next, to assess the effects of UVR, young gametophytes were cultivated under laboratory conditions and exposed to photosynthetically active radiation (PAR) at 80 µmol photons m-2 s-1 and PAR+UVA +UVB for 3 h per day, during three days. The samples were processed for LM and MET to carry out analyses of ultrastructure, growth rates, and content of photosynthetic pigments, as well as, identify carotenoids and evaluate photosynthetic performance. PAR + UVA + UVB caused an increase in cell wall thickness, accumulation of floridean starch grains and changes in the internal organization of the chloroplast. Samples exposed to PAR + UVA + UVB showed a reduction in growth rate of 97%, increased of carotenoids, decreased photosynthetic pigments, in particular, phycoerythrin and allophycocyanin, and a resulting reduction in photosynthetic performance, as observed by the reduction of transport electron (ETR) and ETRmax. Finally, to test the effects of the Cd, young gametophytes of G floridanum were cultured for 7 days with concentrations of 7.5 and 15µM Cd. After the experimental period, LM, confocal and scanning electron microscopy (SEM) were employed toxanalyze growth rates and quantify the content of photosynthetic pigments, carotenoids and chlorophyll a. The treated samples showed a decrease in growth rates, with depigmentation of the thallus and reduction of photosynthetic pigments, resulting in a decrease in the rate of electron transport and autofluorescence of chloroplasts. Furthermore, exposure to Cd proved to be highly toxic, causing chronic photoinhibition, increasing the amount of starch grains, and altering the distribution of protein content in the cytoplasm. It can be concluded that both the Golgi bodies such as the cytoskeletal actin and microtubules are involved in the processes of polarization and germ tube formation during germination tetraspores and that youngs gametophytes of G. floridanum when exposed to both radiation as to the Cd present alterations in metabolism and cellular structure that affect its development

Edoxaban for the Long-Term Therapy of Venous Thromboembolism : Should the Criteria for Dose Reduction be Revised?

Edoxaban is used for venous thromboembolism (VTE) treatment. Real-life data are lacking about its use in long-term therapy. We aimed to assess the efficacy and the safety of edoxaban for long-term VTE treatment in a real-life setting. Patients with VTE included in the egistro nformatizado nfermedad rombombólica (RIETE) registry, receiving edoxaban 60 or 30 mg daily were prospectively followed up to validate the benefit of using different dosages. The main outcome was the composite of VTE recurrences or major bleeding in patients with or without criteria for dose reduction. Multivariable analysis to identify predictors for the composite outcome was performed. From October 2015 to November 2019, 562 patients received edoxaban for long-term therapy. Most (94%) of the 416 patients not meeting criteria for dose reduction received 60 mg daily, and 92 patients meeting criteria (63%) received 30 mg daily. During treatment, two patients developed recurrent VTE, six had major bleeding and nine died (2 from fatal bleeding). Among patients not meeting criteria for dose reduction, those receiving 30 mg daily had a higher rate of the composite event (hazard ratio (HR) 8.37; 95% confidence interval (CI) 1.12-42.4) and a significant higher mortality rate (HR 31.1; 95% CI 4.63-262) than those receiving 60 mg. Among patients meeting criteria for dose reduction, those receiving 60 mg daily had no events, and a nonsignificantly higher mortality rate (HR 5.04; 95% CI 0.54-133) than those receiving 30 mg daily. In conclusion, edoxaban seems to be effective and safe for long-term VTE treatment in real life. Criteria for dose reduction should be reformulated

Increased oxidative damage in carriers of the germline TP53 p.R337H mutation

Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.2060.71, C = 160.560.88, P,0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

DNA damage in homocystinuria: 8-oxo‑,8‑dihydro‑2’-deoxyguanosine levels in cystathionine-β-synthase deficient patients and the in vitro protective effect of N-acetyl‑L‑cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine β-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS‑deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2’- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 μM and 200 μM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria. Keywords: Cystathionine-β-synthase deficiency; oxidative stress; 8-oxo-7,8-dihydro- 2’-deoxyguanosine; homocysteine; DNA damage; N-acetyl-L-cystein

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication