A proteomic approach to guarana seed and pericarp maturation.

Paullinia cupana Kunth var. sorbilis (Mart.) Ducke, the cultivated guarana plant, is native to the Amazon and has been valued for its medicinal, stimulant and energetic properties for centuries. The seeds are the main commercial product of the plant and the source of high amounts of purine alkaloids (caffeine and theobromine) and polyphenols (flavonoids, catechins, and tannins). Proteins involved in the development and maturation of guarana fruits in its native habitat are interesting issues for proteomics. This study presents the proteomic profile of the seed and pericarp of healthy guarana in different maturation stages. Protein contents were higher in the mature seed compared to other stages due to the accumulation of storage proteins - 11S globulins. Proteins selected for identification by mass spectrometry are mostly related to stress responses and defense and this is not unexpected for fast growing and differentiating reproductive tissues.

Peritoneal Submesothelial Stromal Cells Support Hematopoiesis and Differentiate into Osteogenic and Adipogenic Cell Lineages.

The peritoneum is a thin membrane that covers most of the abdominal organs, composed of a monolayer of mesothelial cells and subjacent submesothelial loose connective tissue. Cells from the peritoneal wall are correlated with peritoneal fibrosis and epithelial-to-mesenchymal transition. However, the distinct involvement of mesothelial or submesothelial cells in such phenomena is still not clear. Here, we propose a new strategy to obtain stromal cells from anterior peritoneal wall explant cultures. These cells migrated from peritoneal tissues and proliferated in vitro for 4 weeks as adherent fibroblast-like cells. Optical and electronic microscopy analyses of the fragments revealed a significant submesothelial disorganization. The obtained cells were characterized as cytokeratin- vimentin+ laminin+ α-smooth muscle actin+, suggesting a connective tissue origin. Moreover, at the third passage, these stromal cells were CD90+CD73+CD29+Flk-1+CD45-, a phenotype normally attributed to cells of mesenchymal origin. These cells were able to support hematopoiesis, expressing genes involved in myelopoiesis (SCF, G-CSF, GM-CSF, IL-7 and CXCL-12), and differentiated into osteogenic and adipogenic cell lineages. The methodology demonstrated in this work can be considered an excellent experimental model to understand the physiology of the peritoneal wall in healthy and pathological processes. Moreover, this work shows for the first time that submesothelial stromal cells have properties similar to those of mesenchymal cells from other origins.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Clinical and molecular diagnosis of the skeletal dysplasias associated with mutations in the gene encoding Fibroblast Growth Factor Receptor 3 (FGFR3) in Portugal.

Mutations in the gene that encodes Fibroblast Growth Factor Receptor 3 (FGFR3) are associated with Achondroplasia (MIM 100800), Hypochondroplasia (MIM 146000), Muenke Syndrome (MIM 602849), Thanatophoric Dysplasia (MIM 187600, MIM 187601) and Lacrimo-Auriculo-Dento-Digital Syndrome (MIM 149730).Here we report a clinical and molecular study in a large cohort of 125 Portuguese patients with these skeletal disorders. The identification of the P250R mutation allowed the confirmation of the Muenke Syndrome in 9 out of the 52 cases referred. Two known mutations were found in the Thanatophoric Dysplasia referred cases. No mutations were identified in the LADD syndrome patient. In Achondroplasia and Hypochondroplasia, genetic heterogeneity was present amongst the 70 clinically diagnosed patients with 5 different mutations identified. As in other studies, complex phenotypic heterogeneity amongst patients carrying the same gene defect was observed. In several cases, the new amino acids encoded, as a consequence of mutations, were related to the severity of patients' phenotype. The presence of 10 misdiagnosed cases emphasizes the importance of performing mutation analysis of the hotspot regions responsible for both dysplasias (Ach and Hch). For patients with an unquestionable clinical diagnosis, lacking the most common mutations, a complete screening of FGFR3 is necessary.

Recommendations for Vaccination in Adult Patients with Systemic Inflammatory Rheumatic Diseases from the Portuguese Society of Rheumatology.

BACKGROUND: Serious infections are a major cause of morbidity and mortality in systemic inflammatory rheumatic disease (SIRD) patients. Although vaccination may prevent numerous infections, vaccination uptake rates are low in this group of patients. OBJECTIVES: To develop evidence-based recommendations for vaccination in SIRD patients. METHODS: We searched MEDLINE (until 31 October 2014) and EMBASE (until 14 December 2014) databases, as well as the ACR and EULAR congress abstracts (2011-2014). Patients with any systemic inflammatory rheumatic disease were included and all vaccines were considered. Any safety and efficacy outcomes were admitted. Search results were submitted to title and abstract selection, followed by detailed review of suitable studies. Data were subsequently pooled according to the type of vaccine and the SIRD considered. Results were presented and discussed by a multidisciplinary panel and systematic literature review (SLR)-derived recommendations were voted according to the Delphi method. The level of agreement among rheumatologists was assessed using an online survey. RESULTS: Eight general and seven vaccine-specific recommendations were formulated. Briefly, immunization status should routinely be assessed in all SIRD patients. The National Vaccination Program should be followed and some additional vaccines are recommended. To maximize the efficacy of vaccination, vaccines should preferably be administered 4 weeks before starting immunosuppression or, if possible when disease activity is controlled. Non-live vaccines are safe in SIRD, including immunosuppressed patients. The safety of live attenuated vaccines in immunosuppressed patients deserves further ascertainment, but might be considered in particular situations. DISCUSSION: The present recommendations combine scientific evidence with the multidisciplinary expertise of our taskforce panel and attained desirable agreement among Portuguese rheumatologists. Vaccination recommendations need to be updated on a regular basis, as more scientific data regarding vaccination efficacy and safety, emergent infectious threats, new vaccines as well as new immunomodulatory therapies become available.

Schinzel-Giedion syndrome. A patient with hypothyroidism and diabetes insipidus.

The Schinzel-Giedion is an autosomal recessive syndrome characterized by midface retraction, hypertrichosis, multiple skeletal anomalies, cardiac and renal malformations and mental retardation. We describe a female child with this syndrome and a clinical status complicated by hypernatremic dehydration, hypothyroidism and diabetes insipidus at the age of 10 months.

Robinow syndrome in monozygotic twins with normal stature.

Robinow syndrome was found in two monozygotic twins. We describe the clinical and radiographic manifestations in these patients, both with normal stature and one with omphalocele, with a follow-up of 13 years. Families with Robinow syndrome of both autosomal dominant and recessive inheritance have been reported. We apply the criteria suggested to assign isolated cases to one of the two forms and conclude that autosomal dominant inheritance is more likely.

Biflavonoids from Ouratea multiflora.

A new flavone dimer, 3-hydroxy-4',5,7-trimethoxyflavone-(6-->8")-3"-hydroxy-3"',4"', 5",7"-tetramethoxyflavone, together with amenthoflavone, have been isolated from the leaves of Ouratea multiflora. Its structure was established by spectroscopic methods, including two-dimensional NMR spectroscopy.

[Control of blood transfusion malaria in an endemic and in a non-endemic region in Brazil]. / Controle da malária transfusional em região endêmica e não endêmica do Brasil.

The aim of the present work was to establish appropriate criteria for screening of donor blood from regions with distinct Malaria epidemiological characteristics. Three locations with different screening criteria were studied: São Paulo, SP (with no vectorial transmission), Belém, PA (with low active transmission) and Matupá and Peixoto de Azevedo, MT (with high active transmission). The Malaria parasite--Plasmodium sp--was searched for by "thick film", QBC Test and antigen Immunofluorescence test, and was not detected in any of the samples. There was, however, a great variation in the positivity of anti-plasmodial antibodies, as determined by IIF-IgG anti-P. vivax and -P. falciparum, between accepted donors in the 3 studied locations and between rejected and accepted donors in São Paulo (1.98% accepted, 22.3% rejected--p < 0.01) and Belém (17.2% accepted, 58.3% rejected--p < 0.01). These data endorse the use of the applied clinical and epidemiological screening. In Matupá and Peixoto de Azevedo, where there was no rejected donor, the serological positivity was 80.6%. We, therefore, consider that the Malaria screening in blood banks should follow clinical and epidemiological criteria suitable to each region. The laboratorial screening techniques should then detect either the parasites (thick film/QBC Test or the parasite antigens.

[The effect of the woman's age, the rate of cleavage and embryo quality on obtaining a pregnancy by in-vitro fertilization]. / Repercussão da idade da mulher, da taxa de clivagem e da qualidade embrionária, na obtenção de gravidez por fertilização in vitro.

Multiple factors influence the probability of obtaining a pregnancy through in vitro fertilization (IVF) and embryo transfer (ET). This retrospective study was designed to assess their importance in order to improve prognostic ability and treatment success. 341 consecutive embryo transfer cycles using the same ovarian stimulation protocol were considered and divided in two main groups: 92 cycles in which a clinical pregnancy was achieved and 249 cycles without success. All the embryo transfers were performed in patients from the in vitro fertilization program of the Human Reproductive Unit, Santa Maria Hospital, Lisbon, between January 1991 and December 1993. No significant differences were found between the two groups studied concerning the IVF indications, ovarian response to the stimulation, sperm quality, oocyte maturation and mean number of oocytes retrieved per patient. The women's age was higher in the group which did not achieve a pregnancy, when compared with the pregnant group (p < 0.001), showing a decline of success after the age of 35. Overall oocyte fertilization rate was 88.2% in cycles with pregnancy and 83.5% in cycles without pregnancy (p < 0.02). In the pregnant patients, there was a significantly higher rate of embryo transfers in which all the embryos received had reached at least the four-cell stage at 42-43 hr postinsemination, compared with the non pregnant patients (82% versus 63%, p < 0.001). All the 92 pregnancies originated from transfers of at least one embryo that had undergone two or more mitotic divisions.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacology of biosimilar candidate drugs in rheumatology: a literature review.

OBJECTIVE: To review current evidence concerning pharmacology of biosimilar candidates to be used in rheumatology. METHODS: A PubMed search up to August 2013 was performed using relevant search terms to include all studies assessing pharmacological properties of biosimilar candidates to be used in rheumatology. Data on study characteristics, type of intervention, pharmacokinetics (PK), pharmacodynamics (PD) and bioequivalence ratios was extracted. RESULTS: Of 280 articles screened, 5 fulfilled our inclusion criteria. Two trials, PLANETAS and PLANETRA, compared CT-P13 and infliximab in patients with active ankylosing spondylitis and rheumatoid arthritis, respectively. PK bioequivalence was demonstrated in the phase 1 PLANETAS trial by highly comparable area under the curve (AUC) and maximum drug concentrations (Cmax), whose geometric mean ratios fell between the accepted bioequivalence range of 80-125%. Equivalence in efficacy and safety was demonstrated in the phase 3 PLANETRA trial. Two phase 1 trials comparing etanercept biosimilar candidates TuNEX and HD203 in healthy volunteers showed a high degree of similarity in AUC and Cmax, with respective geometric mean ratios between PK bioequivalence range. The last included trial referred to GP2013, a rituximab biosimilar candidate, which demonstrated PK and PD bioequivalence to reference product in three different dosing regimens in cynomolgus monkeys. CONCLUSION: Infliximab, etanercept and rituximab biosimilar candidates have demonstrated PK bioequivalence in the trials included in this review. CT-P13 has recently been approved for use in the European market and the remaining biosimilar candidates are currently being tested in patients with rheumatoid arthritis.

Portuguese recommendations for the diagnosis and management of gout.

OBJECTIVE: To develop Portuguese evidence-based recommendations for the Diagnosis and Management of Gout. METHODS: As part of the 3e Initiative (Evidence, Expertise and Exchange), a panel of 78 international rheumatologists developed 10 relevant clinical questions which were investigated with systematic literature reviews. MEDLINE, EMBASE, Cochrane CENTRAL and abstracts from 2010-2011 EULAR and ACR meetings were searched. Based on the evidence found in the published literature, rheumatologists from 14 countries developed national recommendations that were merged and voted into multinational recommendations. We present the Portuguese recommendations for the Diagnosis and Management of Gout which were formulated and voted by Delphi method in April 2012, in Lisbon. The level of agreement and potential impact in clinical practice was also assessed. RESULTS: Twelve national recommendations were elaborated from 10 international and 2 national questions. These recommendations addressed the diagnosis of gout; the treatment of acute flares and urate-lowering therapy; monitoring of gout and comorbidity screening; the influence of comorbidities in drug choice; lifestyle; flare prophylaxis; management of tophi and asymptomatic hyperuricaemia; the role of urine alkalinization; and the burden of gout. The level of agreement with the recommendations ranged from 6.8 to 9.0 (mean 7.7) on a 1-10 point visual analogue scale, in which 10 stands for full agreement. CONCLUSION: The 12 Portuguese recommendations for the Diagnosis and Management of Gout were formulated according to the best evidence and endorsed by a panel of 42 rheumatologists, enhancing their validity and practical use in daily clinical practice.

[Rheumatic expression of secondary syphilis]. / Manifestações reumáticas da Sífilis Secundária.

Syphilis is a disease caused by Treponema pallidum infection with protean clinical manifestations. Musculoskeletal complaints are however uncommon and most of the time mild. Occasionally they can dominate the clinical picture and simulate a variety of rheumatic diseases. The authors present the clinical case of a 33-year-old woman who developed a lupus-like syndrome in the postpartum, characterized by polyarthritis, elevated acute phase reactants and positive antinuclear antibodies (ANA). Physical examination revealed a macular non-pruriginous skin rash involving the trunk, upper limbs and palms. The Rapid Plasma Reagin (RPR) and Treponema Pallidum Hemaglutination (TPHA) tests gave a positive result and the patient was diagnosed as secondary syphilis and medicated with 2.4 MU of benzathine penicillin intramuscular weekly for 3 weeks, with complete resolution of clinical signs and ANA negativation. The association of rash and arthritis may occur in several rheumatic diseases but in the presence of palmoplantar involvement, the possibility of syphilis infection should not be overlooked.

[Simultaneous occurrence of neurofibromatosis and tuberous sclerosis, acquired as neo-mutations]. / Ocurrencia simultánea de neurofibromatosis y esclerosis tuberosa, adquiridas como neomutaciones.

INTRODUCTION: Neurofibromatosis type 1 and tuberous sclerosis are two distinct neurocutaneous syndromes that result of a mutation of tumoral suppressor genes, increasing the risk of tumorigenesis. They both have dominant autosomal hereditariness with half of the cases corresponding to new mutations. They are situations rarely associated. CASE REPORT: A boy without any family history of neurocutaneous disorders who had characteristics of both neurofibromatosis and tuberous sclerosis, as cafe-au-lait patches, six greater than 0.5 cm, macrocephaly, optic nerve glioma, focal alterations of the myelin vacuolization of the white matter from both cerebellar hemispheres, brain stem, basal ganglia, characteristic of type 1 neurofibromatosis. He also presented hypopigmentation spots, infantile spasms, and imagiologic findings of cortical areas with altered mielinization on the white matter, left talamo-caudado sulcus calcifications, cortical tubers, Taylor cortical dysplasia, subependimary nodes, characteristically of tuberous sclerosis. The child also had psycho motor development delay. CONCLUSION: The diagnosis of both disorders was confirmed by genetic study. Parents study was negative, so we can confirm the simultaneous occurrence of two new mutations which is unusually rare.

Molecular studies in Portuguese patients with Smith-Lemli-Opitz syndrome and report of three new mutations in DHCR7.

Smith-Lemli-Opitz syndrome (SLO) is an autosomal recessive disorder characterised by craniofacial dysmorphism, mental retardation, multiple congenital anomalies, and increased levels of 7-dehydrocholesterol (7-DHC) in body tissues and fluids. SLO is caused by mutations in the DHCR7 gene which encodes 7-dehydrocholesterol reductase, the last enzyme of cholesterol biosynthesis pathway. In our investigation, we screened 682 dysmorphic/mentally retarded Portuguese patients for abnormal levels of 7-DHC in blood by UV spectrometry. We identified six unrelated patients with SLO (0.87% of total). Mutational analysis of the DHCR7 gene led to the identification of seven distinct mutations, three of which are new (F174S, H301R, and Q98X). The common IVS8-1G > C and T93M variants together with the H301R accounted for 70% of the all SLO alleles in our population. Our findings contribute to the variegate array of pathological changes in the DHCR7 gene among different European populations.