Brassavola tuberculata Hook.: in vitro growth and ex vitro establishment as a function of the micropropagation system and sucrose.

This study examines the in vitro growth and ex vitro establishment of Brassavola tuberculata in relation to the micropropagation system and sucrose concentration employed in the in vitro culture. A completely randomized experimental design was utilized, employing a 2 x 5 factorial arrangement. The experimental period began with seedlings cultivated in vitro for 180 days, which were subsequently transferred to Murashige and Skoog culture media containing sucrose concentrations of 0, 15, 30, 45, or 60 g L-1. The cultures were subjected to two micropropagation systems: conventional and gas exchange. After 90 days of in vitro cultivation, the plants were evaluated, transplanted into a substrate, and placed in a screened nursery for ex vitro cultivation. After 300 days of ex vitro cultivation, the survival and initial characteristics of the plants were assessed. The micropropagation system allowing gas exchange and sucrose concentrations up to 30 g L-1 enhanced the shoot and root growth of in vitro propagated plants. No noticeable anatomical differences were observed after 90 days of in vitro culture among the different sucrose concentrations and micropropagation systems used. In the ex vitro establishment, irrespective of sucrose concentration, the micropropagation system facilitating gas exchange positively influenced all evaluated characteristics.

Structure-activity relationships and mechanism of action of tetragomycin derivatives as inhibitors of Staphylococcus aureus staphyloxanthin biosynthesis.

Despite the main strategy to overcome bacterial resistance has focused on the development of more potent antimicrobial agents, the evolutionary pressure caused by such drugs makes this strategy limited. Molecules that interfere with virulence factors appear as a promising alternative though, as they cause reduced selective pressure. As a matter of fact, staphyloxanthin biosynthesis inhibition (STXBI) has been pursued as promising strategy to reduce S. aureus virulence. Herein, we report the inhibitory profile of 27 tetrangomycin derivatives over staphyloxanthin production. The experimental result showed that naphthoquinone dehydro-α-lapachone (25 - EC50 = 57.29 ± 1.15 µM) and 2-Isopropylnaphtho[2,3-b]furan-4,9-dione (26 EC50 = 82.10 ± 1.09 µM) are the most potent compounds and suggest that hydrogen acceptor groups and lipophilic moieties decorating the naphthoquinone ring are crucial for STXBI. In addition, we present an in situ analysis, through RAMAN spectroscopy, that is inexpensive and might be employed to probe the mechanism of action of staphyloxanthin biosynthesis inhibitors. Therefore, our molecular simplification strategies afforded promising lead compounds for the development of drugs that modulate S. aureus staphyloxanthin biosynthesis.

Liraglutide modulates gut microbiota and reduces NAFLD in obese mice.

Metabolic disorders such as insulin resistance and diabetes are associated with obesity and nonalcoholic fatty liver disease (NAFLD). The aggressive form of a fatty liver disease may progress to cirrhosis and hepatocellular carcinoma. Furthermore, recent studies demonstrated that there is a dysbiosis in the gut microbiota associated with early stages of metabolic disease. Therefore, the identification and repurposing of drugs already used to treat insulin resistance may be an excellent option for other disorders. We evaluated the effect of liraglutide on obesity, NAFLD and gut microbiota modulation in two different animal models of obesity: the ob/ob mice and the high-fat diet (HFD)-fed mice. Liraglutide treatment induced significant weight loss in both obesity models, showed improvements in glycemic parameters and reduced inflammatory cell infiltration in the cecum and the liver. In ob/ob mice, the liraglutide treatment was able to reduce the accumulation of liver fat by 78% and reversed steatosis in the HFD mice. The gut microbiota analysis showed that liraglutide changed the overall composition as well as the relative abundance of weight-relevant phylotypes such as a reduction of Proteobacteria and an increase of Akkermansia muciniphila in the treated HFD group. We show that liraglutide can lead to weight loss and gut microbiota modulations, and is associated with an improvement of NAFLD. Furthermore, by generating a profile of the intestinal microbiota, we compiled a list of potential bacterial targets that may modulate metabolism and induce a metabolic profile that is considered normal or clinically controlled.

Bilateral third-degree burn of the legs: lower limb salvage with dermal regenerative matrix.

Third-degree burns of the lower extremities are among the most difficult burn injuries to treat as they frequently expose bone, tendons or articular surfaces. Coverage with a flap is the ideal treatment, but local tissue is often unavailable, and free flaps require sophisticated equipment and perfect microsurgical technique. We demonstrate a treatment option to obtain a stable cutaneous coverage for this kind of injury, consisting in an association of skin grafts, amniotic membrane and bilaminar dermal matrix templates. This combined treatment proved to be an excellent option to cover a wide area of tibial exposure with low donor site morbidity and good functional and aesthetic results. This shows that artificial dermis is a good alternative for treating bone exposure, especially in patients for whom a classic flap reconstruction is not suitable.

Les brûlures du troisième degré distales des membres inférieurs sont parmi les plus difficiles à traiter en raison de la fréquence des expositions osseuses, tendineuses ou articulaires. Bien que l'utilisation de lambeaux soit le traitement idéal de ces expositions, les tissus adjacents sont souvent inutilisables et les lambeaux libres requièrent un équipement spécifique et une maitrise des techniques microchirurgicales. Nous soumettons une option thérapeutique permettant d'obtenir une couverture cutanée stable pour ce type de brûlures : association de greffe cutanée, de membrane amniotique et de matrice dermique double couche. Ce traitement combiné s'est révélé être un excellent choix thérapeutique pour couvrir de larges expositions tibiales avec peu de morbidité de site donneur et un bon résultat fonctionnel et esthétique. Cela démontre que le derme artificiel peut être une alternative thérapeutique pour les expositions osseuses, surtout lorsque qu'une couverture par lambeau n'est pas possible.

Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi.

UNLABELLED: The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by (1)H and (13)C - nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L. infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to pentamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. CONCLUSION: This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy.

Fitness costs associated with field-evolved resistance to chlorantraniliprole in Plutella xylostella (Lepidoptera: Plutellidae).

Plutella xylostella (L.) is the most important pest of Brassicaceae worldwide, with a recent estimate of US$ 4-5 billion expenditure for the control of this insect. A case of very high resistance of this pest to chlorantraniliprole was recently associated with reduced efficacy in a Brazilian field of Brassica spp. Although diamide resistance has been characterized, the fitness of insects due to such resistance has yet to be examined. Therefore, in this study, biological parameters were assessed in both susceptible and resistant strains of P. xylostella subjected to sublethal chlorantraniliprole concentrations. The field strain showed high resistance to chlorantraniliprole (RR50=27,793-fold), although resistance rapidly decreased in the first generations, showing instability. The exposure of susceptible and resistant larvae to their respective LC1, LC10, and LC25 values led to an increased duration of the larval and pupae phases and reduced weight in both strains; however, no significant differences in pupal viability across the treatments were observed. The resistant insects presented significantly lower larval weight and fecundity and higher larval and pupal periods, hatchability, and male longevity when not exposed to chlorantraniliprole, suggesting a fitness cost associated with resistance. In addition, resistant females showed a significantly higher egg-laying period and longevity at LC25, whereas the males lived longer at LC1. Chlorantraniliprole negatively impacted the biological parameters of both strains tested, although these effects were more relevant to the resistant insects. Resistant P. xylostella showed negative and positive biological trade-offs when compared with the susceptible individuals in both the absence and presence of chlorantraniliprole. Despite the important role that these trade-offs may play in the evolution of resistance to chlorantraniliprole, practical applications still depend on such information as the dominance of fitness costs and resistance.

Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica / Homologous articular chondrocytes implantation in osteochondral defects of dogs: technique and histopathological evaluation standardization

Padronizou-se a metodologia para cultura de condrócitos em cães e avaliou-se seu implante em lesões osteocondrais, utilizando-se a membrana biossintética de celulose (MBC) como revestimento. Dez cães, adultos e clinicamente sadios, foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de 4mm de diâmetro e profundidade foram induzidos no sulco troclear de ambos os membros. MBC foi aplicada na base e na superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados formando o grupo-tratado (GT); os do membro esquerdo, sem implante celular, foram designados grupo-controle (GC). A evolução pós-operatória foi analisada com especial interesse nos processos de reparação da lesão, por meio de histomorfometria e imuno-histoquímica para colágeno tipo II e sulfato de condroitina. A cultura de condrócitos homólogos apresentou alta densidade e taxa de viabilidade. Observou-se integridade do tecido neoformado com a cartilagem adjacente na avaliação histológica, em ambos os grupos. Na imuno-histoquímica, verificou-se predomínio de colágeno tipo II no GT. Morfometricamente, não houve diferença significativa entre o tecido fibroso e o fibrocartilaginoso entre os grupos. A cultura de condrócitos homólogos de cães foi exequível. O tecido neoformado apresentou qualidade discretamente superior associado ao implante homólogo de condrócitos, contudo não promoveu reparação por cartilagem hialina.

The aim of the study is to standardize the methodology to achieve canine chondrocytes culture, and evaluate its implant on osteochondral defects made in the femoral trochlear sulcus of dogs, using the cellulose biosynthetic membrane (CBM) as coating. Ten healthy adult dogs without locomotor disorders were used. All animals were submitted to arthrotomy of stifle joints and defects of four millimeters in diameter x four millimeters deep were done in the femoral trochlear sulcus of both limbs. CBM were applied in the lesion base and surface of all limbs. In the treated group (TG), defects of the right limb were filled with cultivated homologous chondrocytes, and in control group (CG), defects of the left limb were left without cellular implant. Postoperative follow up was done by histomorphometry and Collagen type II and anti-chondroitin sulfate immunohistochemistry. The homologous chondrocytes culture showed high density and viability rate. Upon immunohistochemistry the predominance of type II collagen in extracellular matrix of TG was verified. However, no significant statistical difference was observed between the groups upon histomorphometry analysis of fibrous and fibrocartilaginous tissues. Canine homologous chondrocytes culture was practicable. Neoformed tissue showed slightly higher quality in TG, but without promoting repair by the hyaline cartilage.

Changes in food intake, metabolic parameters and insulin resistance are induced by an isoenergetic, medium-chain fatty acid diet and are associated with modifications in insulin signalling in isolated rat pancreatic islets.

Long-chain fatty acids are capable of inducing alterations in the homoeostasis of glucose-stimulated insulin secretion (GSIS), but the effect of medium-chain fatty acids (MCFA) is poorly elucidated. In the present study, we fed a normoenergetic MCFA diet to male rats from the age of 1 month to the age of 4 months in order to analyse the effect of MCFA on body growth, insulin sensitivity and GSIS. The 45% MCFA substitution of whole fatty acids in the normoenergetic diet impaired whole body growth and resulted in increased body adiposity and hyperinsulinaemia, and reduced insulin-mediated glucose uptake in skeletal muscle. In addition, the isolated pancreatic islets from the MCFA-fed rats showed impaired GSIS and reduced protein kinase Ba (AKT1) protein expression and extracellular signal-related kinase isoforms 1 and 2 (ERK(1/2)) phosphorylation, which were accompanied by increased cellular death. Furthermore, there was a mildly increased cholinergic sensitivity to GSIS. We discuss these findings in further detail, and advocate that they might have a role in the mechanistic pathway leading to the compensatory hyperinsulinaemic status found in this animal model.

Presença de animais associada ao risco de transmissão da leishmaniose visceral em humanos em Belo Horizonte, Minas Gerais / Animal presence and the risk for transmission of visceral leishmaniasis in Belo Horizonte, Brazil

Analisou-se o risco de se contrair leishmaniose visceral (LV) e a presença de animais em residências de Belo Horizonte/MG, em 2006. O estudo de caso-controle foi feito por meio de visitas domiciliares, aplicação de questionário e registro de imagens da residência. A estimativa de risco foi mensurada por comparação de condições de moradia entre dois grupos: 1- constituído por 82 casos humanos de LV ocorridos em 2004 e 2- 164 controles (vizinhos dos casos). Os domicílios pertencentes ao grupo 1 foram os que apresentaram os maiores percentuais de presença de animais. Observou-se, pela análise univariada, que a presença de patos, roedores, pássaros e galinhas aumenta o risco de ocorrer LV em 4,18; 1,81; 1,57; e 1,47 vezes, respectivamente. Para os proprietários de cães, o aumento no risco de contrair LV é equivalente a 2,17 vezes e está relacionado ao número de cães no domicílio. O risco de contrair LV é maior 1,87 vezes para moradores com um cão e 3,36 vezes para moradores com dois cães, quando comparados a pessoas que não possuem esses animais.

This study evaluated the animal presence in dwellings and the risk for transmission of visceral leishmaniasis (VL) in Belo Horizonte, Brazil, 2006. A case-control study was conducted by means of dwelling visits, direct interviews, and image register in the houses. The risk estimates were produced comparing the dwelling condition between two groups: 1) 82 human cases of VL recorded in 2004; and 2) 164 controls (neighbors of the first). The cases presented a higher proportion in the animal presence compared to controls. Using an unconditional logistic regression, it was selected the presence of ducks with an OR of 4.18 (CI 95 percent - 0.74 to 23.32); rodents with an OR of 1.81 (CI 95 percent - 0.96 to 3.39); birds with an OR of 1.56 (CI 95 percent - 0.90 to 2.69), and chicken with an OR of 1.47 (CI 95 percent - 0.74 to 2.90). The owners of dogs were 2.17 more likely to have VL than those who did not have dogs, and this estimate increased with the number of dogs in the house. For those who had only one dog, the OR was 1.87, while for those who owned two dogs at home, the OR increased to 3.36; when compared to people who did not own those animals.

Correlation between clinical signs of depth of anaesthesia and cerebral state index responses in dogs during induction of anaesthesia with propofol.

The cerebral state index (CSI) is used for monitoring EEG and depth of anaesthesia. The objective of this study was to analyse the correlation between ocular reflexes, CSI and estimated propofol plasma concentrations (PropCP) in dogs during induction of anaesthesia with propofol. Fourteen dogs were premedicated with acepromazine 0.05 mg kg(-1) IM. Anaesthesia was induced with a 200 ml h(-1) propofol 1% constant infusion rate until loss of corneal reflex using RugLoop II software with Beths' pharmacokinetic model to estimate PropCp. Palpebral reflex (PR) and the corneal reflex (CR) were tested every 30s and classified as present (+) or absent (-), and eyeball position was registered as rotated ventromedialy (ERV) or centred (EC). Heart rate (HR), mean arterial pressure (MAP) and CSI values were analyzed from baseline before the beginning of propofol infusion (T0) until loss of CR; CSI and PropCp, CSI and anaesthetic planes, and PropCp and anaesthetic planes were compared using correlation analysis. PropCp reached 7.65+/-2.1 microg ml(-1) at the end of the study. CSI values at T0 were 89.2+/-3.8. Based on the observation of ocular reflexes and eyeball position, it was possible to define five anaesthetic planes: A (superficial) to E (deep), being A (PR+/CR+/EC), B (PR+/ERV/CR+), C (PR-/ERV/CR+), D (PR-/EC/CR+) and E (PR-/EC/CR-). There was a significant correlation between PropCp and the anaesthetic planes (R=0,861; P<0.01). No significant correlation was observed between CSI and the anaesthetic planes or between CSI and PropCp. MAP decreased significantly from T0 until loss of corneal reflex (from 98+/-14 mm Hg to 82+/-12 mm Hg); HR did not change significantly (from 101+/-30 bpm to 113+/-16 bpm). The CSI monitoring was not consistent with the clinical observations observed in the different stages of depth anaesthesia. This could limit the use of CSI for monitoring depth of anaesthesia with propofol.

Use of complementary and alternative medicine by Brazilian oncologists.

Complementary and alternative medicine (CAM) is frequently employed by patients with cancer. An extensive survey was conducted among Brazilian cancer physicians to understand their attitude towards CAM. A questionnaire was sent to all 655 members of the Brazilian Cancer Society asking what is their opinion regarding CAM and if they would prescribe any CAM modality for their patients. They were also questioned regarding their degree of awareness of CAM self-administration by their patients. Overall, 119 questionnaires were returned to us (18%). Most oncologists knew at least one type of CAM (96.6%) and 76.7% had previously made use of at least one type of CAM for themselves. We observed that 76 (63.8%) of the oncologists used to ask their patients about CAM utilization and 37.8% described at least one reason to stimulate its use (68.8% as complementary treatment). Only 10% of the oncologists would prescribe at least one type of CAM and this attitude correlated significantly with previous physicians' use of CAM and with being a clinical oncologist as well as with having questioned patients about CAM use. Most oncologists (80.7%) would not indicate the use of CAM, mainly for lack of scientific proof of its efficacy (56.2%). Physicians knew many kinds of CAM and had frequently used some of them themselves, but only a minority of Brazilian oncologists would indicate them. As CAM use is very prevalent in our population, we believe that most of its utilization depends, probably, on patient's own and independent initiatives. However, these results should be viewed with caution because of the low response rate we observed in this study.

Prevalences of hyperhomocysteinemia, unfavorable cholesterol profile and hypertension in European populations.

BACKGROUND: Hyperhomocysteinemia (HHCY) is a risk factor for cardiovascular diseases (CVD). HHCY may interact with hypertension (HTEN) and an unfavorable cholesterol profile (UNFAVCHOL) to alter the risk of CVD. OBJECTIVES: To estimate the prevalences of HHCY (1) isolated and (2) in combination with UNFAVCHOL and/or HTEN in different age categories. To provide information that may improve the screening and treatment of subjects at risk of CVD. DESIGN: Cross-sectional data on 12,541 men and 12,948 women aged 20 + y were used from nine European studies. RESULTS: The prevalence of isolated HHCY was 8.5% in subjects aged 20-40 y, 4.7% in subjects aged 40-60 y and 5.9% in subjects aged over 60 y. When combining all age groups, 5.3% had isolated HHCY and an additional 5.6% had HHCY in combination with HTEN and/or UNFAVCHOL. The combinations of risk factors increased with age and, except for HHCY&UNFAVCHOL, were more prevalent than predicted by chance. Of the young subjects (20-40 y), 24% suffered from one or more of the investigated CVD risk factors. This figure was 75.1% in the old subjects (60+ years). CONCLUSIONS: A substantial number of subjects in selected European populations have HHCY (10.9%). In half of these cases, subjects suffer also from other CVD risk factors like UNFAVCHOL and HTEN. Older people in particular tend to have more than one risk factor. Healthcare professionals should be aware of this when screening and treating older people not only for the conventional CVD risk factors like UNFAVCHOL and HTEN but also HHCY, as this can easily be reduced through increased intake of folic acid via supplement or foods fortified with folic acid.

The PORtromb Project: prothrombin G20210A mutation and venous thromboembolism in young people.

Recently a new identified genetic variant in the 3'-untranslated region of the prothrombin gene (G20210A allele) associated with increased plasma prothrombin levels has been linked to an increased risk of venous thromboembolism (VTE). To determine the importance of this G20210A allele as a causative risk factor for VTE in the general population, we analysed the data of an epidemiologic investigation on thrombophilia in young people, the Oporto Thrombophilia Study (PORtromb). Forty cases (mean age: 27 yr old) with a first episode of VTE under 40 yr old, and 100 healthy subjects, were evaluated. Heterozygosity for the G20210A allele was present in 12.5% of VTE cases and in 5% of controls, indicating a possible marginal increase of VTE risk in carriers of the allele (odds ratio: 2.71: 95% CI 0.74-9.95).

Blood group antigen profile predicted by molecular biology-use of real-time polymerase chain reaction to genotype important KEL, JK,RHD, and RHCE alleles.

The most clinically important blood group systems in transfusion medicine, excluding the ABO system, are the RH, Kell, and Kidd systems. Alloantibodies to antigens of these systems may be produced following blood transfusion or during pregnancy and can result in serious hemolytic transfusion reactions and hemolytic disease of the newborn. We developed rapid and robust techniques for RHD, RHCE, KEL, and JK genotyping with the use of a real-time polymerase chain reaction instrument. Two fluorescence-based methods for the detection of amplification products were used: for KEL1/KEL2, JK1/JK2, and RHE/RHe (exon 5) we used the hybridization probes protocol; for RHC/RHc the analysis was done in sequences of exon 1 for RHC and exon 2 for RHc; and for RHD, analysis was done in sequences of intron 4, exon 7, and exon 4 pseudogene using the SYBR Green I protocol. The genotyping tests were validated with samples from 85 Caucasian Portuguese and 15 Black European blood donors. Complete phenotype-genotype correlations were obtained. The potential use of the presented methods can be predicted in clinical transfusion medicine, allowing appropriate monitoring, early intervention, and improved care. When blood group genotyping techniques are necessary, this methodology is highly competitive for a routine laboratory.

Rapid genotyping of the major alleles at the Duffy (FY) blood group locus using real-time fluorescence polymerase chain reaction.

The Duffy blood group system has clinical importance due to involvement in transfusion reactions and hemolytic disease of the newborn. Recently, the molecular basis of the two alleles, FY*A and FY*B (125G>A), and the mutation situated in the promoter region of the FY gene (-33T>C), have been elucidated. In order to develop an accurate, easy, and rapid genotyping method, we describe a procedure using the LightCycler. Samples from 53 Caucasian Portuguese blood donors and 7 black, healthy, European individuals were phenotyped with commercial antisera. DNA was extracted from blood samples and the relevant sequences were amplified with the same cycling conditions, using real-time polymerase chain reaction. The melting point of the FY*A allele was 63 degrees C and of the FY*B allele, 55 degrees C. The allele without mutation at the promoter region had a melting point at 64 degrees C and the FY*B silent allele at 58 degrees C. The results in Caucasian individuals were similar to those found in European and American populations. When FY genotyping techniques are necessary, the methodology described is preferable to conventional methods as it is reliable, high speed, and uses small volumes, providing a highly competitive technology for use by a routine laboratory.