Unlocking the molecular realm: advanced approaches for identifying clinically and environmentally relevant bacteria

Rapid, effective, and specific identification of clinical and environmental bacterial pathogens is of major importance for their control. Traditionally, bacteria have been identified by phenotypic methods based on morphological, biochemical, and metabolic properties. While these methods are very useful in clinical practice, they have limitations including a poor ability to differentiate within and between species and time-consuming workflows. Newly developed molecular methods can greatly improve the accuracy of taxonomic characterization, identifying specific strains of medical or environmental importance. However, due to high costs and the need for trained professionals, these methods are not yet routine in diagnostic laboratories. Thus, disseminating knowledge on advances in molecular identification techniques is pivotal to make these methodologies accessible. The objective of this work was to review and discuss current molecular techniques for bacteria identification aiming to track and monitor microbial agents in clinical and environmental samples.

Body, metabolic and renal changes following cross-sex estrogen/progestogen therapy in a rodent model simulating its use by transwomen.

PURPOSE: The use of sex steroids by trans people has been of paramount importance regarding body changes during gender transition. The objective of this study was to assess the effects of an injectable steroid combination frequently used by transwomen, namely estradiol enanthate with dihydroxyprogesterone acetophenide (E2EN/DHPA), on blood pressure and metabolic outcomes using an animal model. METHODS: Two-month-old male Wistar rats were orchiectomized or sham-operated and divided into groups: (1) Sham treated with sesame oil vehicle (SG), (2) sham treated with E2EN/DHPA (SP), (3) orchiectomized rats treated with vehicle (OG), and (4) orchiectomized rats treated with E2EN/DHPA (OP), with all groups treated every 10 days for 5 months. We evaluated systolic blood pressure (SBP), body weight (BW), abdominal circumference, nasoanal length (NAL), food and water intake (FI, WI), lipid profile (triglycerides, LDL, and HDL), serum C-reactive protein (CRP), plasma concentrations of urea (URpl) and creatinine (CRpl), 24 h urinary volume (V24 h), sodium and potassium excretion (UNa+, UK+), and proteinuria. RESULTS: E2EN/DHPA administration reduced BW (SP 324.5 ± 31.1; OP 291.7 ± 41.3 g) and NAL (SP 24.5 ± 0.4; OP 24.6 ± 1.0 cm), without changing blood pressure, but increased URpl concentration (SP 55.0 ± 4.8; OP 42.5 ± 8.8 mg/dL) and CRpl (SP 0.47 ± 0.05; OP 0.46 ± 0.04 mg/dL), sodium (SP 3.1 ± 0.8; OP 3.3 ± 0.4 µEq/min/kg), potassium (SP 0.91 ± 0.22; OP 0.94 ± 0.22 µEq/min/kg) excretions and urinary volume (SP 15.5 ± 2.1; OP 16.4 ± 2.9 mL/24 h). CONCLUSION: Cross-sex hormone therapy with E2EN/DHPA significantly modified body characteristics in male rats, producing a feminizing change without altering blood pressure or generating harmful metabolic parameters, but larger translational studies are still needed.

Composição nutricional e valores energéticos determinados com frangos de corte de coprodutos do processamento do etanol de milho / Nutritional composition and energy values determined with broilers of co-products from the processing of corn ethanol

It was determined by the total collection of excreta method, with broilers from 22 to 32 days of age, the coefficients of apparent metabolism of dry matter, crude protein and crude energy and apparent metabolizable energy corrected for zero nitrogen balance (AMEn) of the ethanol co-products of corn: acid oil and dried distillery grains with soluble (DDGS). The DDGS and corn acid oil presented nutritional metabolization coefficients ranging from 43 to 83% and AMEn equal 2393.5 and 7859.2kcal/kg respectively, and may be food alternatives to soybean meal and soybean oil.(AU)

Timed artificial insemination plus heat I: effect of estrus expression scores on pregnancy of cows subjected to progesterone-estradiol-based protocols.

Expression of estrus near timed artificial insemination (TAI) is associated with greater fertility, and estrus detection could improve TAI fertility or direct TAI management, although accurate estrus detection can be difficult and time-consuming using traditional methods. The aim of this study is to evaluate influence of estrus on pregnancy (artificial insemination pregnancy rates (P/AI)) and to validate an alternative method to classify estrus/heat expression using tail chalking (HEATSC) in postpartum Bos indicus cows subjected to TAI in progesterone-estrogen-based protocols. In experiment 1 (Exp. 1), cows (5491) were subjected to visual observation of estrus after progesterone device removal, before TAI, and P/AI was evaluated according to estrus and body condition score (BCS). Cows received a progesterone device and 2 mg estradiol benzoate (EB). After 8 days, the device was removed and 150 µg of d-cloprostenol and 300 IU equine chorionic gonadotrophin was given. Later, animals in Exp. 1 received 1 mg EB and TAI 44 to 48 h. In the Exp. 2 - 3830 cows using similar protocol, received different ovulation inducers: 1 mg EB (n=1624) or 1 mg estradiol cypionate (EC; n=2206) on day 8 (D8). Cows were then marked with chalk, and HEATSC evaluated at TAI on D10 (HEATSC1 - no chalk removal=no estrus expression; HEATSC2 - partial chalk removal=low estrus expression; HEATSC3 - near complete/complete chalk removal=high estrus expression). In Exp. 1, cows showing estrus presented greater P/AI (48.4% v. 40.2%, P<0.05). In Exp. 2, P/AI (HEATSC1 - 40.0%; HEATSC2 - 49.7%; HEATSC3 - 60.9%; P<0.001), and larger follicle timed artificial insemination (LFTAI) (<0.001) varied according to HEATSC. There was no difference in P/AI (P=0.41) or LFTAI (P=0.33) according to ovulation inducer. Cows with greater BCS showed greater P/AI in both experiments (P<0.05). Estrus presence and greater HEATSC improved P/AI, and EC v. EB used promoted differential estrus manifestation (cows showing HEATSC2 and HEATSC3: 79.5% with EB v. 69.98% with EC use, P<0.001), however, with similar P/AI. The use of HEATSC in B. indicus cows subjected to TAI is useful to identify cows with greater estrus expression and consequently improved pregnancy rates in TAI, allowing the cows with low HEATSC to be targeted for additional treatments aimed at improving P/AI.

Repurposing strategies for Chagas disease therapy: the effect of imatinib and derivatives against Trypanosoma cruzi.

Chagas disease (CD) is a neglected parasitic condition endemic in the Americas caused by Trypanosoma cruzi. Patients present an acute phase that may or not be symptomatic, followed by lifelong chronic stage, mostly indeterminate, or with cardiac and/or digestive progressive lesions. Benznidazole (BZ) and nifurtimox are the only drugs approved for treatment but not effective in the late chronic phase and many strains of the parasite are naturally resistant. New alternative therapy is required to address this serious public health issue. Repositioning and combination represent faster, and cheaper trial strategies encouraged for neglected diseases. The effect of imatinib (IMB), a tyrosine kinase inhibitor designed for use in neoplasias, was assessed in vitro on T. cruzi and mammalian host cells. In comparison with BZ, IMB was moderately active against different strains and forms of the parasite. The combination IMB + BZ in fixed-ratio proportions was additive. Novel 14 derivatives of IMB were screened and a 3,2-difluoro-2-phenylacetamide (3e) was as potent as BZ on T. cruzi but had low selectivity index. The results demonstrate the importance of phenotypic assays, encourage the improvement of IMB derivatives to reach selectivity and testify to the use of repurposing and combination in drug screening for CD.

Cervical penetration rates and efficiency of non-surgical embryo recovery in estrous-synchronized Santa Inês ewes after administration of estradiol ester (benzoate or cypionate) in combination with d-cloprostenol and oxytocin.

The effects of estradiol esters, d-cloprostenol and oxytocin on induction of cervical dilation prior to non-surgical embryo recovery in Santa Inês ewes (Days 6-7 estrous cycle) were assessed in this study. In Trial 1, transcervical embryo flushing was performed in estrous-induced ewes administered 37.5 µg of d-cloprostenol i.m. 10 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing with (EB-PGF-OT; n = 13) or without (PGF-OT; n = 11) 1 mg of estradiol benzoate i.m. administered concurrently with d-cloprostenol injection. In Trial 2, the estrous-synchronized animals were treated with 1 mg of estradiol benzoate (EB-PGF-OT; n = 12) or estradiol cypionate (EC-PGF-OT; n = 12) i.m. along with 37.5 µg of d-cloprostenol i.m. 16 h before and 50 IU of oxytocin i.v. 20 min before uterine flushing. In Trial 1, uterine flushing could be accomplished in 38% of ewes in the EB-PGF-OT and 27% those in the PGF-OT (P>0.05) group. Flushing fluid recovery averaged 90% and there were 1.0 ± 1.1 embryos/ewe collected with mean duration of the flushing procedure being ˜36 min. In Trial 2, uterine flushing was accomplished in 78% of ewes in the EB-PGF-OT and 44% of those in the EC-PGF-OT group (P>0.05) with mean flushing fluid recovery rate being 88% and time elapsing to complete flushing being ˜33 min. Within the subsets of animals treated with EB, the percentages of successful transcervical penetrations were 38% compared with 78% in Trials 1 and 2, respectively (i.e., with EB administered 10 h compared with 16 h before uterine flushing: P<0.05). The interval from EB administration to the beginning of transcervical penetration can affect the efficacy of embryo recovery procedures utilizing a combined EB/d-cloprostenol/oxytocin pre-treatment.

Anti-inflammatory and antinociceptive effects of phonophoresis in animal models: a randomized experimental study.

The aim of this study was to evaluate the therapeutic effects of ultrasound (US)-mediated phonophoresis alone or in association with diclofenac diethylammonium (DCF) administered topically in animal models of inflammation. A pre-clinical, prospective, and randomized experimental study of quantitative and qualitative nature was carried out. Phonophoresis was performed using a therapeutic ultrasound apparatus in two distinct models of acute inflammation. Edema was induced by an intraplantar injection of carrageenan and measured by plethysmography. The Hargreaves test was used to evaluate the antinociceptive activity and investigate the action of phonophoresis on tumor necrosis factor (TNF)-α production. A histological analysis with hematoxylin-eosin was used to evaluate tissue repair, and the expression of COX-2 was determined by immunohistochemical analysis. At the peak of inflammatory activity (3 h), treatment with US, US+DCF, and DCF significantly reduced edema formation compared to the control group. Treatment with US+DCF was more effective than treatment with US alone at both analyzed times. In the analysis of the antinociceptive activity, the treatments significantly increased the latency time in response to the thermal stimulus. Histopathological analysis revealed a reduction of the inflammatory infiltrates and immunohistochemistry demonstrated that the association was effective in reducing COX-2 expression compared to the control group. The association of DCF with US produced anti-inflammatory and antinociceptive effects in rat models of inflammation, which may be associated with inhibition of COX-2 and TNF-α production.

Anti-inflammatory and antinociceptive effects of phonophoresis in animal models: a randomized experimental study

The aim of this study was to evaluate the therapeutic effects of ultrasound (US)-mediated phonophoresis alone or in association with diclofenac diethylammonium (DCF) administered topically in animal models of inflammation. A pre-clinical, prospective, and randomized experimental study of quantitative and qualitative nature was carried out. Phonophoresis was performed using a therapeutic ultrasound apparatus in two distinct models of acute inflammation. Edema was induced by an intraplantar injection of carrageenan and measured by plethysmography. The Hargreaves test was used to evaluate the antinociceptive activity and investigate the action of phonophoresis on tumor necrosis factor (TNF)-α production. A histological analysis with hematoxylin-eosin was used to evaluate tissue repair, and the expression of COX-2 was determined by immunohistochemical analysis. At the peak of inflammatory activity (3 h), treatment with US, US+DCF, and DCF significantly reduced edema formation compared to the control group. Treatment with US+DCF was more effective than treatment with US alone at both analyzed times. In the analysis of the antinociceptive activity, the treatments significantly increased the latency time in response to the thermal stimulus. Histopathological analysis revealed a reduction of the inflammatory infiltrates and immunohistochemistry demonstrated that the association was effective in reducing COX-2 expression compared to the control group. The association of DCF with US produced anti-inflammatory and antinociceptive effects in rat models of inflammation, which may be associated with inhibition of COX-2 and TNF-α production.

Estresse oxidativo em cães com doença periodontal: comparação dos biomarcadores plasmáticos e salivares / Oxidative stress in dogs with periodontal disease: comparison of plasmatic and salivary biomarkers

A doença periodontal (DP) é a enfermidade inflamatória mais comum da cavidade oral dos cães. A quantificação de biomarcadores do plasma e da saliva tem sido utilizada para avaliar o estresse oxidativo sistêmico (EOS) e local (EOL) da DP humana. Na DP canina, os mecanismos do estresse oxidativo não estão bem caracterizados e estabelecidos. O objetivo do presente estudo foi investigar a hipótese de que o EOS ocorre na DP canina e de que a saliva pode ser utilizada para avaliar o EOL. Analisou-se, também, a hipótese de que a ativação do metabolismo oxidativo dos neutrófilos contribui para EOS na DP dos cães. Para tal, foram selecionados 20 cães adultos portadores de DP, agrupados de acordo com o grau de lesão: gengivite (n=6), periodontites leve (n=8) e avançada (n=6). O grupo controle foi composto pelos mesmos 20 cães, 30 dias após o tratamento periodontal. Para avaliar o metabolismo oxidativo dos neutrófilos circulantes foi quantificada a produção de superóxido pelo teste de redução do tetrazólio nitroazul (NBT). As concentrações de oxidante total (TOC) e de espécies reativas ao ácido tiobartbitúrico (TBARS) no plasma foram quantificadas para avaliar o EOS. Para a avaliação do estresse oxidativo local, foi quantificado o TOC salivar e a concentração dos principais antioxidantes da saliva (albumina, ácido úrico e bilirrubina total). O EOS na DP foi confirmado pelo aumento da produção de superóxido dos neutrófilos circulantes, TOC e TBARS plasmático. Foi possível quantificar todos os biomarcadores na saliva de cães, porém nenhum foi capaz de expressar o EOL da DP canina. Esta é uma das primeiras evidências de que o EOS ocorre em cães com DP e que a ativação do metabolismo oxidativo dos neutrófilos pode contribuir para desequilíbrio entre antioxidantes e oxidantes. Este estudo ressalta a importância da higiene bucal dos cães para a prevenção da DP e de lesões degenerativas crônicas de diversos tecidos causadas pelo EOS.(AU)

Periodontal disease (PD) is the most common inflammatory disease of the oral cavity of dogs. Quantitation of plasma and salivary biomarkers have been used to assess the systemic oxidative stress (SOS) and local (LOS) of human PD. In canine PD, oxidative stress mechanisms are not well characterized and established. Our objective was to investigate the hypothesis that SOS occurs in dog PD and saliva can be used to evaluate the LOS. We also investigated the hypothesis that the activation of neutrophil oxidative metabolism contributes to SOS in dog SD. For this purpose, 20 adult dogs were selected PD patients, grouped according to the degree of injury: gingivitis (n=6), light periodontitis (n=8) and advanced periodontitis (n=6). The control group was composed of the same 20 dogs, 30 days after periodontal treatment. To assess oxidative metabolism of circulating neutrophils superoxide production was measured by test nitroblue tetrazolium reduction (NBT). The total oxidant concentrations (TOC) and reactive species to tiobartbitúrico acid (TBARS) in plasma were quantified to evaluate SOS. For the evaluation of local oxidative stress were quantified salivary TOC and concentration of the main antioxidant in saliva (albumin, uric acid, and total bilirubin). EOS in dogs with PD was confirmed by increased superoxide production of circulating neutrophils, TOC, and plasma TBARS. It was possible to quantify all the biomarkers in the saliva of dogs, but none was able to express the LOS canine PD. This is the first evidence that SOS occurs in dogs with PD and that activation of the oxidative metabolism of neutrophils may contribute to an imbalance between oxidants and antioxidants. This study highlights the importance of oral hygiene of dogs to prevent PD and chronic degenerative lesions of various tissues caused by SOS.(AU)

One Hundred Consecutive Liver Transplants Using Institutes Georges Lopez-1 Preservation Solution: Outcomes and Prognostic Factors.

BACKGROUND: There are only 4 prior studies reporting on outcomes of liver transplantation (LT) using Institutes Georges Lopez-1 (IGL-1) preservation solution. Detection of negative predictors of LT using IGL-1 may help finding strategies to protect selected recipients at higher risk of graft failure and death. METHODS: Review of all consecutive adult patients who underwent a first whole-graft LT using IGL-1 at authors' institution from 2013 to 2016. Primary end point was graft failure within the first 90 postoperative days (PODs). Graft losses due to any cause (including all deaths with a functioning graft) were recorded as graft failures. RESULTS: Of all 100 patients included in this study, 37 were women; median age was 58 years (range 18-71). There were 12 graft losses during the first 90 PODs (including 3 cases of primary nonfunction of the liver allograft), and 10 of the 12 graft losses occurred on first 30 PODs. All 12 patients who experienced graft loss (including 1 patient who underwent liver retransplantation) died within the first 90 PODs. Of the total 100 patients, 14 experienced biliary complications. Univariate analysis revealed prolonged warm ischemic time (WIT) as the only predictor of 90-day graft failure (odds ratio = 23.5, confidence interval = 1.29-430.18, P = .03). The cutoff by receiver operating characteristic curve for WIT was 38 minutes (area under the curve = 0.70). Positive predictive value for WIT >38 minutes was 94.3%. CONCLUSIONS: LT using IGL-1 can be performed safely. Similar to prior reports on LT using other preservation solutions, prolonged WIT was associated with adverse outcomes.

Parâmetros nutricionais e estabilidade aeróbia de silagens de cereais de inverno submetidas a diferentes regimes de corte no estádio vegetativo / Cutting system in vegetative stage alters nutritional parameters and aerobic stability of Winter cereals silage

O uso de regimes de corte em estádio vegetativo para os cereais de inverno é interessante quando se busca intensificar a produção de alimento volumoso para ruminantes. No entanto, é necessário se averiguar o impacto desse manejo sobre as características das silagens resultantes. Dessa forma, o objetivo do experimento foi avaliar o valor nutricional e a estabilidade aeróbia de silagens de cereais de inverno, submetidas a regimes de corte no estádio vegetativo, prévio ao corte para ensilagem. Os cereais utilizados foram trigo (Triticum aestivum cv. BRS Gralha Azul), cevada (Hordeum vulgare cv. BRS Brau), aveia-branca (Avena sativa cv. URS Guará), aveia- preta (Avena strigosa cv. Embrapa 139) e triticale (X Triticosecale cv. IPR 11), associados a três regimes de corte: sem cortes prévios (silagem exclusiva); um corte; e com dois cortes em estádio vegetativo e subsequente produção de silagem. Na avaliação nutricional, a cevada apresentou valores baixos de FDA (311,7 e 375,3g kg de MS-1) e altos de NDT (569,2 e 533,4g kg de MS-1) para os sistemas com um e dois cortes, respectivamente. Na avaliação da estabilidade aeróbia, os materiais foram bastante estáveis após a abertura dos silos, com exceção da aveia-branca e da aveia-preta, no regime sem cortes. As silagens de triticale e de cevada apresentaram as maiores estabilidades nutricionais com o aumento no número de cortes.(AU)

The use of cutting regimes in vegetative stage for winter cereals is interesting when it seeks to intensify the forage production for ruminants. However, it is necessary to ascertain the impact of this management on the characteristics of the resulting silage. Thus, the aim of the experiment was to evaluate the nutritional quality and aerobic stability of winter cereal silages submitted to cutting regimes in vegetative stage, before ensiling. The materials used were: wheat (Triticum aestivum cv. BRS Gralha Azul); barley (Hordeum vulgare cv. BRS Brau); white oat (Avena sativa cv. URS Guará); black oat (Avena strigosa cv. Embrapa 139) and triticale (X Triticosecale cv IPR 11); associated with three management strategies: without previous cuts (Only silage), one cut and two cuts in vegetative and posterior silage making. Barley owned low values of ADF (311.7 and 375.3g kg de DM-1) and high TDN (569.2 and 533.4g kg de DM-1) for one and two-cut system, respectively. For aerobic stability, materials were quite stable after opening the silos, with the exception of black and white oat in management without cuts. Triticale and barley silage had the highest nutritional stability with the increase in the number of cuts.(AU)

Doctors' awareness concerning primary immunodeficiencies in Brazil.

BACKGROUND: PIDs are a heterogeneous group of genetic illnesses, and delay in their diagnosis is thought to be caused by a lack of awareness among physicians concerning PIDs. The latter is what we aimed to evaluate in Brazil. METHODS: Physicians working at general hospitals all over the country were asked to complete a 14-item questionnaire. One of the questions described 25 clinical situations that could be associated with PIDs and a score was created based on percentages of appropriate answers. RESULTS: A total of 4026 physicians participated in the study: 1628 paediatricians (40.4%), 1436 clinicians (35.7%), and 962 surgeons (23.9%). About 67% of the physicians had learned about PIDs in medical school or residency training, 84.6% evaluated patients who frequently took antibiotics, but only 40.3% of them participated in the immunological evaluation of these patients. Seventy-seven percent of the participating physicians were not familiar with the warning signs for PIDs. The mean score of correct answers for the 25 clinical situations was 48.08% (±16.06). Only 18.3% of the paediatricians, 7.4% of the clinicians, and 5.8% of the surgeons answered at least 2/3 of these situations appropriately. CONCLUSIONS: There is a lack of medical awareness concerning PIDs, even among paediatricians, who have been targeted with PID educational programmes in recent years in Brazil. An increase in awareness with regard to these disorders within the medical community is an important step towards improving recognition and treatment of PIDs.

Bioactive compounds and antioxidant capacity of buriti (Mauritia flexuosa L.f.) from the Cerrado and Amazon biomes.

Pulp of buriti palms (Mauritia flexuosa L.f.) from two Brazilian regions had their phenolic and carotenoid contents and antioxidant capacity evaluated through different methods (ABTS, DPPH, FRAP and ORAC). Buriti pulp from the Cerrado presented higher phenolic levels (435.08 mg AGE 100 g(-1)) and elevated antioxidant capacity in all tests. The fruits from the Amazon region had a higher total carotenoid content (52.86 mg 100 g(-1)). Carotenoid profile has confirmed ß-carotene predominance in both regions studied. The buritis' region of origin influenced bioactive compound contents and antioxidant capacity of the fruits analyzed. A significant positive correlation (r ⩽ 0.95; p<0.05) was observed between total phenolics and antioxidant capacity through the different methods applied. Results showed promising prospects for the future exploitation of buriti fruits as a potential source of carotenoids and natural antioxidants.

First report of a de novo mutation at SLC20A2 in a patient with brain calcification.

Primary familial brain calcification (PFBC) is identified by mineralization of the basal ganglia and other brain regions in the absence of known causes. The condition is often inherited in an autosomal dominant pattern and can manifest itself clinically with neuropsychiatric symptoms such as Parkinsonism, headaches, psychosis, and mood swings. Mutations in the SLC20A2 gene account for ~40% of inherited cases, and this gene encodes an inorganic phosphate transporter (PiT-2), a transmembrane protein associated with Pi homeostasis. The p.Y386X mutation in SLC20A2 was identified in a patient who presented migraines, brain calcification, and mild but chronic hypovitaminosis D. SLC20A2 c.1158C > G single-nucleotide heterozygous mutation results in a premature stop codon and a putative truncated protein of 385 amino acids. Proband parents do not present the mutation, which is also not present in major public SNP databases, suggesting a de novo sporadic trait. This study describes for the first time a de novo SLC20A2 mutation in a PFBC patient with migraine and mild hypovitaminosis D. This data further reinforces the pathogenic role of SLC20A2 mutations as causal factors in PFBC physiopathology.

Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and no apparent genotype-phenotype correlation: an appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families.

BACKGROUND: We previously identified a four-generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. The entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. METHODS: A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. The entire coding region of the RET gene was investigated by direct sequencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. RESULTS: An additional p.C634Y germline mutation in the RET gene was identified in the reported family. The double mutation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program developed at our institution, we additionally report the combination of these two mutations (p.C634Y/p.Y791F) in the RET gene in four other unrelated families. The overall penetrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, respectively. CONCLUSION: Our data emphasises that a comprehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atypical clinical course of the disease.

Application of five prognostic survival scores to primary myelofibrosis in 62 Brazilian patients.

Primary myelofibrosis (PM) is a Philadelphia-negative clonal hematopoietic stem cell disorder characterized by intense reactive changes of bone marrow stroma with collagen fibrosis, osteosclerosis and angiogenesis. PM usually affects elderly people, and approximately half of the patients present JAK2V617F mutation. PM clinical course varies from 1 to 30 years, evolving from asymptomatic into progressive bone marrow failure, symptomatic splenomegaly or acute leukemia in 10-20 % of cases. PM risk stratification is based on parameters predicting survival, and several attempts have been made to identify clinical and laboratory features that could predict PM patient survival. This study applied five prognostic scores: Dupriez, Cervantes, Mayo, IPSS and DIPSS-Plus in 62 Brazilians patients from three centers, and compared their relevance and clinical usefulness considering the scores' parameters, fibrosis, JAK2V617F mutation, splenomegaly, hepatomegaly and treatment. According to the Cervantes, Dupriez and Mayo scores, most patients were stratified into low-risk group. However, when IPSS and DIPSS-Plus were applied, most patients were classified into an intermediate range, being low risk in only 11 and 13 % of patients, respectively. Overall survival at 4 years was 84 %. The Cervantes score was the only one that remained significantly associated with survival in a multivariate analysis. In conclusion, the Cervantes score remains important to the prognostication of PM.

Role of the extracellular matrix in variations of invasive pathways in lung cancers.

Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas (SqCC), is the massive degradation of the extracellular matrix. The remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan synthases (HAS), E-cadherin adhesion molecules, and the transforming growth factor ß (TGF-ß) may favor invasion, cellular motility, and proliferation. We examined HAase proteins (Hyal), HAS, E-cadherin, and TGF-ß profiles in lung AD subtypes and SqCC obtained from smokers and non-smokers. Fifty-six patients, median age 64 years, who underwent lobectomy for AD (N = 31) and SqCC (N = 25) were included in the study. HAS-1, -2 and -3, and Hyal-1 and -3 were significantly more expressed by tumor cells than normal and stroma cells (P < 0.01). When stratified according to histologic types, HAS-3 and Hyal-1 immunoreactivity was significantly increased in tumor cells of AD (P = 0.01) and stroma of SqCC (P = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS-3 immunoreactivity in tumor cells (P < 0.01). Stroma cells of SqCC from non-smokers presented a significant association with HAS-3 (P < 0.01). Hyal, HAS, E-cadherin, and TGF-ß modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. HAases in resected AD and SqCC were strongly related to the prognosis. Therefore, our findings suggest that strategies aimed at preventing high HAS-3 and Hyal-1 synthesis, or local responses to low TGF-ß and E-cadherin, may have a greater impact in lung cancer prognosis.

Role of the extracellular matrix in variations of invasive pathways in lung cancers

Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas (SqCC), is the massive degradation of the extracellular matrix. The remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan synthases (HAS), E-cadherin adhesion molecules, and the transforming growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation. We examined HAase proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and non-smokers. Fifty-six patients, median age 64 years, who underwent lobectomy for AD (N = 31) and SqCC (N = 25) were included in the study. HAS-1, -2 and -3, and Hyal-1 and -3 were significantly more expressed by tumor cells than normal and stroma cells (P < 0.01). When stratified according to histologic types, HAS-3 and Hyal-1 immunoreactivity was significantly increased in tumor cells of AD (P = 0.01) and stroma of SqCC (P = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS-3 immunoreactivity in tumor cells (P < 0.01). Stroma cells of SqCC from non-smokers presented a significant association with HAS-3 (P < 0.01). Hyal, HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. HAases in resected AD and SqCC were strongly related to the prognosis. Therefore, our findings suggest that strategies aimed at preventing high HAS-3 and Hyal-1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis.

Association of CD28 and CTLA-4 gene polymorphisms with aggressive periodontitis in Brazilians.

OBJECTIVE: Susceptibility to and severity of periodontal disease is influenced by gene polymorphisms related to the immune response. Co-stimulatory molecules, such as CD28 and CTLA-4, are critical in the development of such responses. Our hypothesis is that polymorphisms in genes that code for these molecules may be associated with periodontitis. The aim of the study was to investigate the association between +17 (T/C) CD28 and +49 (A/G) CTLA-4 gene polymorphisms and periodontitis in Brazilians. MATERIALS AND METHODS: Genomic DNA was obtained from oral swabs of 424 individuals categorized into three groups (control group, aggressive, and chronic periodontitis) considering clinical parameters such as probing depth and clinical attachment loss. The genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There was an association between the T(-) genotype of the CD28 polymorphism and aggressive periodontitis (P = 0.04). Moreover, the A(+) genotype for CTLA-4 was associated with greater clinical attachment loss in non-smokers with aggressive periodontitis (P = 0.006, OR = 16.25, CI = 2.25-117.11). CONCLUSIONS: These findings show that T(-) in CD28 + 17 (T/C) and the A(+) in CTLA-4 +49 (A/G) genotypes are associated with susceptibility to aggressive periodontal disease. Thus, our study highlights these polymorphisms as potential genetic susceptibility markers of periodontitis in Brazilians.