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.

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.

Cattleya walkeriana Gardner (Orchidaceae) propagation: culture medium, sealing system and irradiance.

This study aimed to evaluate the influence of culture media, irradiance, and sealing system on the in vitro and ex vitro growth of Cattleya walkeriana Gardner. We used MS medium as culture medium, supplemented with 30 g L-1 of sucrose and solidified with 7.0 g L-1 of bacteriological agar. This medium served as a control, while for the other treatments we supplemented the media as follows: 2) MS with 150 g L-1 of banana pulp = P150; 3) MS with 300 g L-1 of banana pulp = P300; 4) MS with 150 g L-1 of banana peel = PE150; and 5) MS with 300 g L-1 of banana peel = PE300. The irradiances were provided by 3000K LED lamps: 86 µmol m-2 s-1 (Irradiance-1) and 128 µmol m-2 s-1 (Irradiance-2) and the conventional sealing (CSS) and sealing systems that allow gas exchange (GESS). After 120 (in vitro) and 180 days (ex vitro) of cultivation, we evaluated them for pseudobulb (PN), leaf (LN) and root number (RN), plant height (PH), pseudobulb diameter (PD), longest leaf (LL) and root length (RL), fresh mass (TFM) and survival (%SURV). There was a significant interaction for all the variables analyzed. The CM x SS double interaction was significant for PH, LL, and RL. The CM x I x SS interaction was significant for PN, LN, RN, PD, TFM, and %SURV traits of C. walkeriana grown in vitro. There was a significant interaction between CM x I x SS for all C. walkeriana traits evaluated in ex vitro culture. Using the medium with up to 150 g L-1 of banana pulp combined with Irradiance-2 and CSS provided the highest values for in vitro plant growth. However, prior cultivation in MS medium, Irradiance-1, and CSS provided the greatest survival and establishment of this species plants in ex vitro culture.

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.

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.

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.

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.

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.

Comparison of the simplex and Powell methods with a weighted response function for the optimization of FIA systems.

An experimental comparison between the relative performances of the Powell and simplex methods for the optimization of a Flow Injection Analysis (FIA) system for the determination of nitrite in water is reported. An evaluation of the advantages of using a weighted linear combination of two variables (related to sensitivity and sample throughput rate) as the response function in the guidance of the optimization procedures towards different practical requirements is also included. Both methods proved to be effective for the optimization, none having shown definite advantages over the other. The use of a weighted response function in these optimization methods proved to be useful for assessing the versatility of FIA systems.

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.

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.

An efficient medium for the isolation of Trichomonas foetus.

Trials were conducted to compare a modification of a commercial medium with the 2 media currently used at the Veterinary Research Institute, Onderstepoort, for the isolation of Trichomonas foetus. An antifungal agent was also added to the medium. Results obtained proved the modified medium to be more reliable for the isolation of T. foetus than the other 2, and the addition of antifungal more efficient in the control of contaminants.

The use of filter paper discs impregnated with thionin acetate, basic fuchsin and thionin blue in the identification of Brucella species.

Filter paper discs impregnated with solutions containing 0.25, 0.5 and 1 mg/milliliter of thionin acetate, 0.75 and 1.5 mg/milliliter of basic fuchsin and 0.5 mg/milliliter of thionin blue were used in the typing of Brucella species. All the strains used reacted as expected, proving this new technique to be reliable in the identification of Brucella species. The method is less expensive and the results easier to interpret than those obtained with methods previously used.

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle.

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.

A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls.

Seven batches of 25% water-phase, oil-in-water vaccine were prepared from whole cultures of Tritrichomonas foetus. Two inoculations were given, spaced 6 weeks apart, to virgin heifers and infected bulls. A significant reduction (P less than 0.01) in the duration of infection in vaccinated heifers was seen when they were challenged by being bred to a bull infected with the same isolate as that contained in the vaccine. Only 1/12 vaccinated heifers were pregnant 4.5 months after the end of the breeding season compared to 2/12 in the control group. The vaccine, therefore, has no practical advantage. Vaccine was supplied to 2,724 bulls on properties where the infection was present. From these bulls, 110 reliable results were obtained, where bulls had been infected, been inoculated and tested 1 month later. No curative effect was demonstrable with 69/110 (62.7%) bulls, remaining infected after the course of inoculations. There was also no difference between vaccine batches or between bulls of different ages. Further work on improving the vaccine is indicated. Three media suitable for the culture of T. foetus are described in detail.

The use of sensitivity discs in the identification of Campylobacter species.

Filter paper discs were impregnated with a solution containing 20 mg of triphenyltetrazolium chloride per millilitre, and used in the typing of catalase-positive Campylobacter species. Also used were filter paper discs impregnated with cephalothin at 30 micrograms/ml, 60 micrograms/ml and 3 mg/ml and nalidixic acid at the same concentrations, as well as commercially available discs containing 30 micrograms of, respectively, cephalothin and nalidixic acid. Results obtained proved the technique to be reliable and easier to interpret than previously used methods, and laboratory prepared filter paper discs compared favourably with commercial discs.

The isolation and serology of Brucella melitensis in a flock of goats in central RSA.

Brucella melitensis biotype 1 was isolated in pure culture from the lungs, liver, spleen, kidney, stomach contents, abomasum and brain of an aborted caprine (Boer goat) foetus in the district of Cullinan near Pretoria. The 18 does and 1 ram in the flock of Boer goates were examined serologically by means of the complement fixation (CF) test, using Brucella abortus antigen. Six weeks later they were examined again, using B. abortus as well as B. melitensis biotype 1 antigens. No significant differences were found between the 2 CF tests using B. abortus antigen, or between the results obtained by using the B. abortus and B. melitensis antigens. Twelve goats, showing CF antibody titres, were slaughtered and examined bacteriologically. No relationship was found between the serological and bacteriological results.

The persistence of serological reactions following heifer vaccination with Brucella abortus strain 19 on brucellosis-free farms.

Of 392 heifers vaccinated between 4 and 9.5 months of age and tested at 22 months or older, 3 (0.77%) had titres in the complement fixation test of between 30 and 98 international units per millilitre. The results confirm that persistent serological reactions can be a problem even in brucellosis-free herds. To reduce the incidence of these problem reactors, it is suggested that: in clean herds the age of inoculation be reduced to 3 months of age; the age at first testing be delayed as long as possible, but be done before the heifers are 4 months pregnant, c) consideration be given to reducing the vaccine dose for all heifers from the present 4 to 12 x 10(10) to between 3 x 10(8) and 3 x 10(9) and syringes used for strain 19 vaccination be exclusively reserved for this purpose to prevent the possibility of antigenic residues acting as a booster when used for other vaccinations.

[Oxygen free-radicals in medicine (2)]. / Radicais livres do oxigénio em medicina (2. parte).

Free radicals are highly reactive molecules, and therefore transient, which have an odd number of electrons and are generated in vivo as byproducts of normal metabolism. In this review we survey basic concepts on the chemistry of oxygen free radicals, their cellular sources and the reactions they can undergo. We also discuss the cellular defenses against free radicals induced damage. The disfunction induced by free radicals may thus be a major component of several pathological conditions. The critical role played by free radicals in ageing, carcinogenesis, reperfusion injury and respiratory distress is reviewed.

[Oxygen free radicals in medicine (I)]. / Radicais livres do oxigénio em medicina (1. Parte).

Free radicals are highly reactive molecules, and therefore transient, which have an odd number of electrons and are generated in vivo as byproducts of normal metabolism. In this review we survey basic concepts on the chemistry of oxygen free radicals, their cellular sources and the reactions they can undergo. We also discuss the cellular defenses against free radicals induced damage. The disfunction induced by free radicals may thus be a major component of several pathological conditions. The critical role played by free radicals in ageing, carcinogenesis, reperfusion injury and and respiratory distress is reviewed.