Imbalanced IL10/TGF-ß production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis.

BACKGROUND: Human T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-ß. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines. METHODS: Asymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-ß-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells. RESULTS: Total frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group. CONCLUSIONS: A higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-ß may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Eventos adversos em crianças submetidas a cirurgias orofaciais / Adverse events in children submitted to orofacial surgery / Eventos adversos en niños sometidos a cirugía orofacial

Objetivo: Identificar os eventos adversos no pós-operatório imediato de queiloplastia e/ou palatoplastia em crianças e comparar os eventos identificados aos notificados ao Núcleo de Segurança do Paciente. Métodos: Estudo descritivo, retrospectivo e quantitativo, realizado em um hospital público e terciário brasileiro. Os dados foram coletados por meio da descrição nos registros de enfermagem e comparados aos notificados ao Núcleo de Segurança do Paciente, referente a junho e dezembro de 2019. Os resultados foram submetidos a análise estatística descritiva. Resultados: A amostra constou de 203 crianças, das quais 51% (n=103) apresentaram evento adverso. Foram identificados 176 eventos adversos, de 8 tipos, com prevalência da laringite pós-extubação (n=50; 28%), edema de língua (n=34; 19%) e lesão de comissura labial (n=25; 14%). Destes, apenas 5% (n=9) foram notificados ao Núcleo de Segurança do Paciente. Conclusão: Os eventos adversos prevalentes se relacionaram a cavidade oral e tecidos adjacentes, e a subnotificação foi expressiva. (AU)

Objective: To identify adverse events in the immediate postoperative period of cheiloplasty and/or palatoplasty in children and compare the identified events to those notified to the Patient Safety Center. Methods: Descriptive, retrospective and quantitative study, carried out in a Brazilian public and tertiary hospital. Data were collected through descriptions in nursing records and compared to those notified to the Patient Safety Center, referring to June and December 2019. The results were subjected to descriptive statistical analysis. Results: The sample consisted of 203 children, of which 51% (n=103) had an adverse event. A total of 176 adverse events of 8 types were identified, with prevalence of post-extubation laryngitis (n=50; 28%), tongue edema (n=34; 19%) and labral commissure lesion (n=25; 14%). Of these, only 5% (n=9) were notified to the Patient Safety Center. Conclusion: The prevalent adverse events were related to the oral cavity and adjacent tissues, and underreporting was significant. (AU)

Objetivo: Identificar eventos adversos en el postoperatorio inmediato de queiloplastia y/o palatoplastia en niños y comparar los eventos identificados con los notificados al Centro de Seguridad del Paciente. Métodos: Estudio descriptivo, retrospectivo y cuantitativo, realizado en un hospital público y terciario brasileño. Los datos se recolectaron mediante descripciones en registros de enfermería y se compararon con los notificados al Centro de Seguridad del Paciente, referidos a junio y diciembre de 2019. Los resultados fueron sometidos a análisis estadístico descriptivo. Resultados: La muestra estuvo conformada por 203 niños, de los cuales el 51% (n = 103) tuvo un evento adverso. Se identificaron un total de 176 eventos adversos de 8 tipos, con prevalencia de laringitis posextubación (n=50; 28%), edema de lengua (n=34; 19%) y lesión de la comisura del labrum (n=25; 14%). De estos, solo el 5% (n=9) fueron notificados al Centro de Seguridad del Paciente. Conclusion: Los eventos adversos prevalentes se relacionaron con la cavidad bucal y los tejidos adyacentes y el subregistro fue significativo. (AU)

Jóvenes universitarios de género masculino y el uso del preservativo / Jovens universitários do gênero masculino e a utilização do preservativo / Young male university students and condom use

Objetivo: Analizar el uso sistemático del condón por estudiantes universitarios varones. Método: Estudio transversal realizado en dos instituciones de educación superior de Río de Janeiro, con 661 estudiantes sexualmente activos de 18 a 29 años que respondieron un cuestionario. Los datos fueran analizados con el software Statistical Package for Social Sciences mediante estadística descriptiva e inferencial, con la aplicación de la prueba chi-cuadrado de Pearson. Resultados: Los datos muestran que los jóvenes son solteros (61,98%); heterosexuales (87,37%); no usan condones con parejas fijas (45,40%) o ocasionales (23,55%). El estado civil, la orientación sexual, las creencias religiosas, el consumo de alcohol y otras drogas antes de la última relación sexual, la percepción de riesgo y antecedentes de infecciones de transmisión sexual son un factor determinante para el uso del condón (valor de p <0,05). Conclusión: Considerando las características de la juventud y los factores socioculturales involucrados, se observa que los estudiantes asumen conductas de riesgo ante las infecciones de transmisión sexual con el uso no sistemático del condón. (AU)

Objective: to analyze systematic condom use by male university students. Method: A cross-sectional study carried out in two higher education institutions from Rio de Janeiro, with 661 sexually active students aged 18-29, who answered a questionnaire. The data were analyzed using the Statistical Package for Social Sciences software through descriptive and inferential statistics, with application of Pearson's chi-square test. Results: The data show that young people are single (61.98%), heterosexuals (87.37%) and that they do not use condoms with steady (45.40%) or occasional (23.55%) partners. Marital status, sexual orientation, religious belief, use of alcohol and other drugs before the last sexual relationship, risk perception and past history of sexually transmitted infections are determining factors for condom use (p-value<0.05). Conclusion: Considering the characteristics inherent to youth and the sociocultural factors involved, it is noted that the students assume risk behaviors in the face of sexually transmitted infections with non-systematic condom use. (AU)

Objetivo: Analisar o uso sistemático do preservativo por universitários do gênero masculino. Método: Estudo transversal realizado em duas instituições de ensino superior no Rio de Janeiro, com 661 estudantes de 18-29 anos, sexualmente ativos, que responderam um questionário. Os dados foram analisados com emprego do softwareStatistical Package for the Social Sciences através da estatística descritiva e inferencial, com a aplicação do teste qui-quadrado de Pearson. Resultados: Dados demonstram que os jovens são solteiros (61,98%); heterossexuais (87,37%); não usam preservativo com parcerias fixas (45,40%) ou eventuais (23,55%). A situação conjugal, orientação sexual, crença religiosa, uso de álcool e outras drogas antes da última relação sexual, percepção de risco e história pregressa de infecção sexualmente transmissível são fatores determinante para uso do preservativo (p-valor<0,05). Conclusão: Considerando as características próprias da juventude e fatores socioculturais envolvidos, nota-se que os estudantes assumem comportamentos de risco frente às infecções sexualmente transmissíveis com a utilização não sistemática do preservativo. (AU)

Dynamics of hospitalizations and in-hospital deaths from COVID-19 in northeast Brazil: a retrospective analysis based on the circulation of SARS-CoV-2 variants and vaccination coverage.

This study investigated the dynamics of hospitalizations and in-hospital deaths from coronavirus disease 2019 (COVID-19) throughout the pandemic in northeast Brazil, the Brazilian region with the worst socioeconomic indicators. In total, 141,445 cases, 8,213 hospital admissions, and 1,644 in-hospital deaths from COVID-19 were registered from March 14, 2020 to February 5, 2022. The overall rates of hospitalization and in-hospital deaths were 5.8% and 20.0%, respectively. The hospitalization and death rates significantly decreased over time, which may have been related to progress in vaccination. During the spread of the Gamma variant (January to June 2021), most hospitalized individuals were young adults, and approximately 40% of deaths occurred in this age group. During the predominance of Delta (July to December 2021), over 75% of deaths occurred among the elderly and unvaccinated or partially vaccinated individuals. This rate decreased to 42.3% during the transmission of the Omicron variant (January to February 2022), during which 34.6% of deaths were recorded among fully vaccinated individuals (2 doses) and 23.1% among those who received full vaccination and a booster. The Omicron-driven third wave was associated with a rise in the proportion of deaths among vaccinated individuals, especially among those who had not received a booster dose.

A compositional link between rocky exoplanets and their host stars.

Stars and planets both form by accreting material from a surrounding disk. Because they grow from the same material, theory predicts that there should be a relationship between their compositions. In this study, we search for a compositional link between rocky exoplanets and their host stars. We estimate the iron-mass fraction of rocky exoplanets from their masses and radii and compare it with the compositions of their host stars, which we assume reflect the compositions of the protoplanetary disks. We find a correlation (but not a 1:1 relationship) between these two quantities, with a slope of >4, which we interpret as being attributable to planet formation processes. Super-Earths and super-Mercuries appear to be distinct populations with differing compositions, implying differences in their formation processes.

Tos4 mediates gene expression homeostasis through interaction with HDAC complexes independently of H3K56 acetylation.

Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3's well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.

Glutamine and cystine-enriched diets modulate aquaporins gene expression in the small intestine of piglets.

The regulation of glycerol permeability in the gastrointestinal tract is crucial to control fat deposition, lipolysis and gluconeogenesis. Knowing that the amino acid glutamine is a physiological regulator of gluconeogenesis, whereas cystine promotes adiposity, herein we investigated the effects of dietary supplementation with glutamine and cystine on the serum biochemical parameters of piglets fed on amino acid-enriched diets, as well as on the transcriptional profile of membrane water and glycerol channels aquaporins (AQPs) in the ileum portion of the small intestine and its impact on intestinal permeability. Twenty male piglets with an initial body weight of 8.8 ± 0.89 kg were allocated to four dietary treatments (n = 5) and received, during a four week-period, a basal diet without supplementation (control) or supplemented with 8 kg/ton of glutamine (Gln), cystine (Cys) or the combination of the two amino acids in equal proportions (Gln + Cys). Most biochemical parameters were found improved in piglets fed Gln and Cys diet. mRNA levels of AQP3 were found predominant over the others. Both amino acids, individually or combined, were responsible for a consistent downregulation of AQP1, AQP7 and AQP10, without impacting on water permeability. Conversely, Cys enriched diet upregulated AQP3 enhancing basolateral membranes glycerol permeability and downregulating glycerol kinase (GK) of intestinal cells. Altogether, our data reveal that amino acids dietary supplementation can modulate intestinal AQPs expression and unveil AQP3 as a promising target for adipogenesis regulation.

Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.

DM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor's regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.

Haplotypic characterization of BRCA1 c.5266dupC, the prevailing mutation in Brazilian hereditary breast/ovarian cancer.

Specific pathogenic mutations associated with breast cancer development can vary between ethnical groups. One example is BRCA1 c.5266dupC that was first described as a founder mutation in the Ashkenazi Jewish population, but was later also found in other populations. In Brazil, this mutation corresponds to 20% of pathogenic BRCA1 variants reported. Our objective was to investigate the haplotype component of a group of Brazilian families who inherited c.5266dupC in the BRCA1 gene and to verify the ancestry contribution from European, African, and Amerindian origins. Fourteen probands carrying c.5266dupC and 16 relatives (carriers and non-carriers) were investigated. The same haplotype was observed segregating within all the families analyzed, revealing no recombinants in a region of 0.68 Mb. Ancestry analysis demonstrated that the European component was predominant among probands. The BRCA1 c.5266dupC analysis indicates that there was a founder effect in the Brazilian population.

Does the stretching intensity matter when targeting a range of motion gains? a randomized trial

Abstract Aims: To verify the effects of flexibility training conducted at different intensities in young adults. Methods: Twenty-one (21) young adults of both genders with no history of surgery, fracture, and/or rheumatic diseases in the lower limbs and hip, were randomly assigned to low intensity (LI) or high intensity (HI) stretching groups. Two researchers were assigned to evaluate the active knee extension range of motion (ROM) of the volunteers and two other researchers were responsible for the training program. The training consisted of a single exercise for the hamstring muscles (biceps femoris, semimembranosus, semitendinosus). Each session consisted of three repetitions of passive static stretching, maintained for the 60s each, with 30s interval between them, and performed three times a week for four weeks. The stretching intensity was based on the Numerical Verbal Scale, the LI group maintained the intensity between 1 and 2, while the HI group between 9 and 10. Three ROM evaluations were performed pre-intervention, after the 6th session and at the end of the 12th session. Results: No difference was observed between the groups that underwent either high- or low-intensity programs. Both groups achieved gains in flexibility after four weeks of training. Conclusions: The study demonstrated that both high- and low-intensity stretching exercises are effective for ROM and there were no differences between them. Therefore, the intensity can be defined by the preference of the therapist or patient.

Toxicological analysis of ceramic building materials - Tiles and glasses - Obtained from post-treated bottom ashes.

In Italy, the production of bottom ash from waste incineration was estimated as 1.6 million tons/year, corresponding to 30% of the total input waste. The bottom ash is mainly formed by SiO2, Al2O3, CaO, Na2O and low amount of heavy metals, therefore it cannot be considered a 'non-hazardous' waste. In this context, the aim of this work was to determine the effectiveness of the sintering and vitrification techniques to turn bottom ash into an inert ceramic or glass matrix using toxicological tests. The bottom ash from a municipal solid waste facility was ground and used in ceramic tile and glass compositions. After sintering of the ceramic tiles and melting of the glass compositions, the samples were characterized by leachability and toxicological analyzes. Living organisms were used in the toxicological tests, Escherichia coli and Staphylococcus aureus (Agar Diffusion Test), Artemia sp. (Acute Toxicity Test) and Lactuca sativa (germination) and the results were compared with the plasmid DNA test. Regarding the leachability results, the ceramic tile samples showed a concentration of Cu slightly above the limit determined by the D.M. 5/4/2006 directive and, therefore, could not be considered an inert material. Regarding the toxicological tests, the bottom ash alone is mutagenic, but this effect is avoided once the ash is immobilized into the glasses and ceramic tiles, as demonstrated by the results reported in this study.

Microencapsulation of sacha inchi oil (Plukenetia volubilis L.) using complex coacervation: Formation and structural characterization.

In this study, sacha inchi oil (SIO) (Plukenetia volubilis L.) was microencapsulated via complex coacervation of ovalbumin (OVA) and sodium alginate (AL), and the microcapsule properties were characterized. The omega-3 content in the SIO was evaluated after in vitro gastric simulation and microencapsulation. The coacervate complex between OVA and AL was evaluated based on electrostatic interactions and developed for use as a wall material via the SIO microencapsulation process. The best mass ratio for the biopolymers (OVA:AL) was 4:1 at pH 3.8, and the complex exhibited a thermal resistance at 189.86 °C. The SIO microcapsules showed a high encapsulation efficiency of approximately 94.12% in the ratio (OVA:AL) of 1:1. Furthermore, microencapsulated SIO presented resistance under gastric conditions with a low release of acyl (ω-3) units. These results demonstrate that it is possible to use OVA:AL as encapsulating agents to protect bioactive compounds and to improve the thermal behavior of microcapsules.

From yeast to humans: Understanding the biology of DNA Damage Response (DDR) kinases.

The DNA Damage Response (DDR) is a complex network of biological processes that protect cells from accumulating aberrant DNA structures, thereby maintaining genomic stability and, as a consequence, preventing the development of cancer and other diseases. The DDR pathway is coordinated by a signaling cascade mediated by the PI3K-like kinases (PIKK) ATM and ATR and by their downstream kinases CHK2 and CHK1, respectively. Together, these kinases regulate several aspects of the cellular program in response to genomic stress. Much of our understanding of these kinases came from studies performed in the 1990s using yeast as a model organism. The purpose of this review is to present a historical perspective on the discovery of the DDR kinases in yeast and the importance of this model for the identification and functional understanding of their mammalian orthologues.

Case Report: Strongyloides stercoralis Hyperinfection in a Patient with HTLV-1: An Infection with Filariform and Rhabditiform Larvae, Eggs, and Free-Living Adult Females Output.

Strongyloides stercoralis is the main etiological agent of human strongyloidiasis. Severe strongyloidiasis is commonly associated to alcoholism, corticostereoid use, and human T cell lymphotropic virus type 1 (HTLV-1) coinfection. Herein, we report a case of a 13-year-old boy coinfected with S. stercoralis and HTLV-1, excreting several parasitic forms in the stool. The parasitological examination of his feces showed a large amount of filariform (about 3,000 larvae per gram of feces) and rhabditiform larvae (about 2,000 larvae per gram of feces). In addition, free-living adult females (about 50 parasites per gram of feces) and eggs (about 60 eggs per gram of feces) were detected. The main laboratory findings pointed to high immunoglobulin E (IgE) levels (228 UI/mL) and eosinophila (11.6%). The patient was treated with three courses of ivermectin (200 µg/kg twice, 2 weeks apart), achieving the parasitological cure. An increase of about 19 times in interleucin (IL)-17 level was observed following the parasitological cure, in addition to a decrease in the white blood cell, eosinophil counts, and IgE levels. This is the first case report, to our knowledge, in which an S. stercoralis adult free-living female was described in human feces and where an increase in IL-17 levels after Strongyloides treatment in a HTLV-1 coinfected individual was observed. This finding raises the need for further studies about IL-17 immunomodulation in S. stercoralis and HTLV-1 coinfected patients.

Prognostic impact of micro-rna expression in breast cancer: systematic review / Impacto prognóstico da expressão de micrornas no câncer de mama: revisão sistemática

Breast cancer is an important health problem worldwide and the identification of new prognostic markers is important in establishing the best treatment for each patient. MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression and that can be useful biomarkers for prognosis in breast cancer. The objective of this systematic review was to investigate tumor miRNA expression potentially associated with the prognostic factors of breast carcinomas. The search was done in the PubMed database; 1457 articles were initially found and 20 studies were included in the review. MiRNA-21 and miRNA-200b were the most commonly investigated in breast cancer prognosis. Lymph node metastasis was associated with the hyperexpression of miRNA-211, miRNA-301a and miRNA-370 and also associated with the hypoexpression of miRNA-124, miRNA-127, miRNA-129-5p, miRNA199-5p, miRNA-206, miRNA-218 and miRNA-339-5p. Distant metastasis was associated with miRNA-204 hypoexpression. Tumor size was associated with hyperexpression of miRNA-21 and miRNA-301a and also to the hypoexpression of miRNA-29b and miRNA129-5p. Lower survival rates were associated with the hyperexpression of miRNA-21, miRNA-301a and microRNA-711, and hypoexpression of miRNA-15a, miRNA-29b, miRNA-124, miRNA-129-5p, miRNA 199b -5p, miRNA-200b, miRNA-204, miRNA-206 and miRNA-218. On the other hand, higher survival rates were associated with the hyperexpression of miRNA-339-5p and miRNA-127 and also to the hypoexpression of miRNA-210. The results of this review emphasize the need to validate these findings in additional studies

O câncer de mama é um importante problema de saúde em todo o mundo e a identificação de novos marcadores prognósticos é necessária para estabelecer o melhor tratamento para cada paciente. MicroRNAs (miRNAs) são RNAs não codificadores reguladores da expressão gênica que têm sido evidenciados como biomarcadores úteis no prognóstico do câncer de mama. O objetivo desta revisão sistemática foi verificar o papel da expressão de miRNAs tumorais associados aos fatores prognósticos dos carcinomas de mama. A busca de estudos foi feita no banco de dados PubMed; 1.457 artigos foram inicialmente encontrados e 20 estudos foram incluídos na revisão. MiRNA-21 e miRNA-200b foram os mais comumente investigados em relação aoprognóstico do câncer de mama. A presença de metástase linfonodal foi significativamente associada à hiperexpressão de miRNA-211, miRNA-301a e miRNA-370 e também associada à hipoexpressão de miRNA-124, miRNA-127, miRNA-129-5p, miRNA199-5p, miRNA-206, miRNA-218 e miRNA- 339-5p. Metástase a distância foi associada à hipoexpressão de miRNA-204. O tamanho do tumor foi associado à hiperexpressão de miRNA-21 e miRNA-301a e também à hipoexpressão de miRNA-29b e miRNA129-5p. Em relação à sobrevida global, menores taxas de sobrevida foram associadas à hiperexpressão de miRNA-21, miRNA-301a e microRNA-711 e à hipoexpressão de miRNA- 15a, miRNA-29b, miRNA-124, miRNA-129-5p, miRNA 199b-5p, miRNA-200b, miRNA-204, miRNA-206 e miRNA-218. Por outro lado, maiores taxas de sobrevida foram associadas à hiperexpressão de miRNA-339-5p e miRNA-127 e também à hipoexpressão de miRNA-210. Os resultados desta revisão enfatizam a necessidade de validar esses achados em estudos adicionais

Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

Biomarcadores na dermatite atópica / Biomarkers in atopic dermatitis

O objetivo deste estudo é descrever os achados de artigos recentemente publicados a respeito dos avanços diagnósticos, prognósticos e terapêuticos sobre o uso de biomarcadores na dermatite atópica. Foi realizada revisão bibliográfica de 19 artigos publicados no Journal of Allergy and Clinical Immunology e no The New England Journal of Medicine. Atualmente, mais de 1 bilhão de pessoas têm algum tipo de doença alérgica, entre as quais a dermatite atópica é altamente prevalente. Por mais que possa ser diagnosticada de forma clínica através dos critérios de Hanifin e Rajka, estes são avaliador-dependentes, havendo grande variação dos resultados. Da mesma forma, a dosagem de IgE não é específica. Assim, tais métodos não são precisos para o diagnóstico, aumentando a importância da descoberta de novos marcadores mais fidedignos. Os biomarcadores são características biológicas quantificáveis que fornecem medidas objetivas do estado de saúde ou doença. Eles têm potencial para estratificação de risco, detecção precoce, identificação do tratamento, monitorização de resposta e prevenção da progressão para marcha atópica. Os avanços tecnológicos permitem aos clínicos determinar um grande número de biomarcadores através de fluidos corporais, o que resultará em uma melhor caracterização e estratificação dos pacientes com dermatite atópica, bem como acarretará medidas objetivas da resposta terapêutica e melhores comparações entre tratamentos correntes e novas terapias.

The objective of this study is to describe the findings of recently published articles on the diagnostic, prognostic and therapeutic advances in the use of biomarkers in atopic dermatitis. Nineteen articles published in the Journal of Allergy and Clinical Immunology and in The New England Journal of Medicine were reviewed. Currently, more than 1 billion people have some type of allergic disease; among such diseases, atopic dermatitis is highly prevalent. Even though atopic dermatitis can be diagnosed clinically using the Hanifin and Rajka criteria, diagnosis is examiner-dependent, leading to major variations in results. Likewise, IgE dosage is non-specific. Thus, these methods are not accurate for diagnosis, highlighting the importance of finding new, more reliable markers. Biomarkers are quantifiable biological characteristics that provide objective measures of health status or disease. They have a potential for risk stratification, early detection, treatment identification, response monitoring and prevention of progression to the atopic march. Technological advances allow clinicians to determine a large number of biomarkers through body fluids, which will result in a better characterization and stratification of patients with atopic dermatitis and lead to objective measures of treatment response and better comparisons between current and new therapies.

Oxidative stress in patients with type 2 diabetes mellitus treated with metformin / Estresse oxidativo em pacientes com diabetes mellitus tipo 2 em tratamento com metformina

AIMS: To evaluate oxidative stress parameters in patients with type 2 diabetes mellitus treated with metformin, relating these values to its side effects, plasma levels, glycemic control, diabetic complications, lipid profile, and the influence of pharmacotherapeutic follow-up. METHODS: Patients with type 2 diabetes mellitus, on metformin and in pharmacotherapeutic follow-up for four months, were evaluated. The pharmacotherapeutic follow-up consisted in providing information and answering patients' questions about medication and disease. In addition, administration times, dosages, and presence or absence of side effects related to the use of metformin were verified. Glycemic and lipid profile, oxidative stress (superoxide dismutase and malondialdehyde) and plasma metformin were evaluated. Pearson's correlation and Spearman's correlation were performed to evaluate the relationship between the variables at the beginning of the study. The independent t-test and Mann-Whitney U test were used to assess the difference between the groups with and without diabetic complications. The range of values between the beginning and end of the study was evaluated using Student's t-test or Wilcoxon U test. The significance level was set at 5%. RESULTS: The initial sample consisted of 49 patients aged 59±9 years with a body mass index of 29.8±5.1 kg/m2 , who have had diabetes for a median time of 36 months (interquartile range of 1-240) and have been on metformin for a median time of 36 months (interquartile range of 1-180). Twenty-five patients left the study between the second and fourth meetings. Malondialdehyde levels differed between before and after pharmacotherapeutic follow-up, being positively correlated with blood glucose, glycohemoglobin, and triglyceride level, and negatively correlated with metformin and superoxide dismutase. Blood glucose, glycohemoglobin, and malondialdehyde levels increased, whereas metformin levels decreased in the group with diabetic complications, and there was a correlation between malondialdehyde and the number of diabetic complications per patient. CONCLUSIONS: In this sample of patients with type 2 diabetes mellitus treated with metformin, oxidative stress was more pronounced in those with poor glycemic control and diabetic complications.

OBJETIVOS: Avaliar parâmetros de estresse oxidativo em pacientes com diabetes mellitus tipo 2 em uso de metformina, relacionando estes valores a seus efeitos adversos, níveis plasmáticos, controle glicêmico, complicações diabéticas, perfil lipídico, e a influência do acompanhamento farmacoterapêutico. MÉTODOS: Foram avaliados pacientes com diabetes mellitus tipo 2, em uso de metformina, em acompanhamento farmacoterapêutico por quatro meses. O acompanhamento farmacoterapêutico consistiu na prestação de informações e no esclarecimento de dúvidas dos pacientes sobre a medicação e a doença. Além disto, foram verificados os horários, as doses e a presença ou não de efeitos adversos relacionados ao uso de metformina. Foram avaliados perfil glicêmico e lipídico, estresse oxidativo (superóxido dismutase e malondialdeído) e metformina plasmática. Foram realizados os testes de correlação de Pearson e de Spearman para avaliar as relações entre as variáveis no início do estudo. Para testar a diferença entre os grupos com e sem complicações diabéticas, foram utilizados o t-teste independente ou o teste U de MannWhitney. A gama de valores entre o início e o final do estudo foi avaliada utilizando o teste t de Student ou o teste de Wilcoxon U. Foi adotado um nível de significância de 5%. RESULTADOS: A amostra inicial foi composta por 49 pacientes com idade de 59±9 anos e índice de massa corporal de 29,8±5,1 kg/m2 , com diabetes por uma mediana de tempo de 36 (intervalo interquartil 1-240) meses e em uso de metformina há uma mediana de 36 (intervalo interquartil 1-180) meses. Vinte e cinco pacientes deixaram o estudo entre a segunda e a quarta reunião. Os níveis de malondialdeído diferiram entre antes e após o acompanhamento farmacoterapêutico, correlacionando-se positivamente com glicemia, glicohemoglobina e triglicerídeos e negativamente com metformina e superóxido dismutase. Encontrou-se elevação da glicemia, glicohemoglobina e malondialdeído, e diminuição da metformina no grupo com complicações diabéticas, e foi identificada correlação entre malondialdeído e o número de complicações diabéticas por paciente. CONCLUSÕES: Nesta amostra de pacientes com diabetes mellitus tipo 2 em tratamento com metformina, o estresse oxidativo foi mais pronunciado nos que apresentavam pior controle glicêmico e complicações diabéticas.

Detecção de Variantes nos genes MSH2, MSH6, MLH1 e PMS2 utilizando Sequenciamento de Nova Geração / [Detection of Variants in the MSH2, MSH6, MLH1 and PMS2 using Nova Sequencing Generation]

A Síndrome de Lynch (SL) é uma herança autossômica dominante de alta penetrância, que atinge aproximadamente 3% dos pacientes com câncer colorretal. Os indivíduos acometidos apresentam mutações germinativas em um dos genes responsáveis pelo reparo de DNA por mal pareamento (Mismatch Repair- MMR): MSH2, MLH1, MSH6 ou PMS2. Por serem genes longos, a identificação de mutações nestes genes se torna demorada e com custo elevado. Desta maneira, o projeto teve como objetivo padronizar uma estratégia de identificação de variantes que permitisse reduzir os custos e tempo de rastreamento molecular dos genes MMR. Para isso, foram padronizadas 16 reações de PCRs-multiplex para os genes MSH2, MLH1 e MSH6 e 5 reações de PCRs de longo alcance para o gene PMS2. Estes produtos foram sequenciados utilizando a tecnologia de Nova Geração (NGS), através do equipamento HiSeq2500. A estratégia foi validada através do sequenciamento pelo método de Sanger dos genes de MMR em 66 pacientes, provenientes de quatro centros distintos do Brasil (INCARJ, HCPA- RS, HJUBB- PA e ACCAM- SP), e que preencheram os critérios de Bethesda para SL. As profundidades médias de cobertura obtidas para os genes MSH2, MSH6, MLH1 e PMS2 foram de 7.988, 36.313, 11.899 e 4.772 vezes, respectivamente. Foram identificadas 98 alterações em éxons e íntrons dos quatro genes, sendo que 25 eram patogênicas ou VUS (7 em MSH2, 5 em MSH6, 12 em MLH1 e 1 em PMS2) e foram encontradas em 32 pacientes. A estratégia padronizada foi eficaz na identificação de variantes dos genes MMR, permitiu reduzir em três vezes o número de reações de PCR realizadas por amostra e em 2,15 vezes o custo de rastreamento molecular para os quatro genes MMR. Dos fragmentos sequenciados, 3,1% apresentaram profundidade média de cobertura <30X e não foram eficientes na detecção de sítios variáveis nos genes MMR

Lynch Syndrome (LS) is an autosomal dominant inheritance of high penetrance that affects approximately 3% of the cases of colorectal cancer. Individuals affected with this syndrome inherit germline mutations in one of the genes responsible for the Mismatch Repair: MSH2, MLH1, MSH6 or PMS2. These large numbers of genes turns mutations' identification time consuming and costly. The project aimed to establish a molecular screening method in order to optimize the cost-effectiveness for screening these genes. We performed 16 Multiplex PCRs for MSH2, MSH6 and MLH1 genes and 5 Long-Range PCRs for PMS2 gene, coupled with Next Generation Sequencing (NGS) technologies. Our strategy was validated by the screening of sixty-six patients who filled Bethesda criteria for LS from different hospitals of Brazil (INCA-RJ, HCPA-RS, HJUBB-PA and ACCAM-SP). The depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-eight alterations were found in exons and introns regions for the four MMR genes. From this, 25 were pathogenic or VUS found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). The strategy was efficient to identify genetic changes at the MMR genes, since allowed to reduce to 3 times the number of PCR reactions performed per patient and to reduce in 2,15 times the costs to molecular screening of the four MMR genes. Approximately 3% of the amplicons sequenced had a depth of coverage <30X and were not efficient in variation detection at MMR genes

Detection of CALR Mutation in Clonal and Nonclonal Hematologic Diseases Using Fragment Analysis and Next-Generation Sequencing.

OBJECTIVES: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. METHODS: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases. CALR screening was primarily performed using fragment analysis polymerase chain reaction, then next-generation sequencing and Sanger sequencing. RESULTS: Among the 18 patients diagnosed with clonal diseases, one had acute myeloid leukemia (positive for trisomy 8), and 17 had myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), primary myelofibrosis (PMF), and polycythemia vera (PV). Patients with CML were positive for the BCR-ABL1 fusion. Ten patients were positive for JAK2 (PMF, n = 1; ET, n = 2; PV, n = 7), and three were CALR positive (ET, n = 1; PMF, n = 2). Patients diagnosed with a nonclonal disease were negative for JAK2, BCR-ABL, and CALR mutations. CONCLUSIONS: Screening for CALR mutations is essential in BCR-ABL-negative MPNs since it not only provides valuable diagnostic and prognostic information but also identifies potential treatment targets. Since this study describes the importance of screening for known and novel biomarkers, we described in detail three methods that could be easily integrated into a clinical laboratory.