SYBR safeTM efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safeTM, a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safeTM. For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safeTM. In conclusion, SYBR safeTM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

Novel and highly informative Capsicum SSR markers and their cross-species transferability.

This study was undertaken primarily to develop new simple sequence repeat (SSR) markers for Capsicum. As part of this project aimed at broadening the use of molecular tools in Capsicum breeding, two genomic libraries enriched for AG/TC repeat sequences were constructed for Capsicum annuum. A total of 475 DNA clones were sequenced from both libraries and 144 SSR markers were tested on cultivated and wild species of Capsicum. Forty-five SSR markers were randomly selected to genotype a panel of 48 accessions of the Capsicum germplasm bank. The number of alleles per locus ranged from 2 to 11, with an average of 6 alleles. The polymorphism information content was on average 0.60, ranging from 0.20 to 0.83. The cross-species transferability to seven cultivated and wild Capsicum species was tested with a set of 91 SSR markers. We found that a high proportion of the loci produced amplicons in all species tested. C. frutescens had the highest number of transferable markers, whereas the wild species had the lowest. Our results indicate that the new markers can be readily used in genetic analyses of Capsicum.

Modelling zinc changes at the hippocampal mossy fiber synaptic cleft.

Zinc, a transition metal existing in very high concentrations in the hippocampal mossy fibers from CA3 area, is assumed to be co-released with glutamate and to have a neuromodulatory role at the corresponding synapses. The synaptic action of zinc is determined both by the spatiotemporal characteristics of the zinc release process and by the kinetics of zinc binding to sites located in the cleft area, as well as by their concentrations. This work addresses total, free and complexed zinc concentration changes, in an individual synaptic cleft, following single, short and long periods of evoked zinc release. The results estimate the magnitude and time course of the concentrations of zinc complexes, assuming that the dynamics of the release processes are similar to those of glutamate. It is also considered that, for the cleft zinc concentrations used in the model (≤ 1 µM), there is no postsynaptic zinc entry. For this reason, all released zinc ends up being reuptaken in a process that is several orders of magnitude slower than that of release and has thus a much smaller amplitude. The time derivative of the total zinc concentration in the cleft is represented by the difference between two alpha functions, corresponding to the released and uptaken components. These include specific parameters that were chosen assuming zinc and glutamate co-release, with similar time courses. The peak amplitudes of free zinc in the cleft were selected based on previously reported experimental cleft zinc concentration changes evoked by single and multiple stimulation protocols. The results suggest that following a low amount of zinc release, similar to that associated with one or a few stimuli, zinc clearance is mainly mediated by zinc binding to the high-affinity sites on the NMDA receptors and to the low-affinity sites on the highly abundant GLAST glutamate transporters. In the case of higher zinc release brought about by a larger group of stimuli, most zinc binding occurs essentially to the GLAST transporters, having the corresponding zinc complex a maximum concentration that is more than one order of magnitude larger than that for the high and low affinity NMDA sites. The other zinc complexes considered in the model, namely those formed with sites on the AMPA receptors, calcium and KATP channels and with ATP molecules, have much smaller contributions to the synaptic zinc clearance.

Comparação social, insatisfação corporal e comportamento alimentar em jovens adultos / Social comparison, body dissatisfaction and eating behavior in young adults

O objetivo do presente estudo foi comparar o comportamento de comparação social (CCS) de jovensadultos, segundo a insatisfação corporal (IC) e o comportamento alimentar inadequado (CAI).Participaram 563 sujeitos (276 mulheres) entre 1 8 e 35 anos de idade. Teste U de Mann-Whitney foiutilizado para comparar os CCS segundo a IC e o CAI. Foi identificada diferença para oscomportamentos de comparação social entre o grupo de sujeitos satisfeitos e insatisfeitos, e também paraos grupos com e sem comportamentos alimentares inadequados, do sexo feminino. Conclui-se que ocomportamento de comparação social está associado à insatisfação corporal e aos comportamentosalimentares inadequados de mulheres, o mesmo não ocorrendo para os homens(AU)

ESR1 polymorphisms and statin therapy: a sex-specific approach.

Lipid-lowering therapy has shown a high degree of variability in clinical response and there is evidence that the variability in drug response between individuals is due to genetic factors. Thirteen single nucleotide polymorphisms (SNPs) within the ESR1 gene were evaluated with basal lipid and lipoprotein levels, as well as response to lipid-lowering therapy, in 495 hypercholesterolemic individuals of European descent receiving simvastatin or atorvastatin. Significant associations were detected between rs4870061 (P=0.040, corrected P-value (PC)=0.440), rs1801132 (P=0.002, PC=0.022) and the SNP rs3020314 (P=0.013, PC=0.143) with triglyceride (TG) baseline levels. The rs4870061 was also associated with high-density lipoprotein cholesterol (HDL-C) baseline levels (P=0.045, PC=0.495). Regarding statin efficacy, rs2234693 C/C was associated with greater HDL-C increase (P=0.037; PC=0.407) and rs3798577 T allele was associated with greater total cholesterol (TC) reduction (P=0.019; PC=0.209) and greater TG reduction (P=0.026; PC=0.286). These associations suggest that ESR1 polymorphisms are in part responsible for the TC, HDL-C and TG variation levels and this effect may be sex-specific.

JAK2 46/1 haplotype is associated with JAK2 V617F--positive myeloproliferative neoplasms in Brazilian patients.

INTRODUCTION: This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. METHODS: The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. RESULTS: The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially those that were JAK2 V617F positive, than in the control population. We also compared the 46/1 haplotype status in each MPN disease entity, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPNu with controls. The G allele frequency relative to controls was significantly enriched in patients with PV and ET, but not in those with PMF and MPNu. PV and ET patients especially, all of whom had the JAK2 V617F mutation, showed significant excess of the G allele. The frequency of JAK2 V617F mutation was associated with elevated hematological parameters, but when we analyze the occurrence of the mutation and the presence of the G allele, just the high hemoglobin was significantly. CONCLUSION: In agreement with previous reports, JAK2 46/1 haplotype for JAK2 V617F was associated with cMPN positive in Brazilian patients.

Eating behaviours in youths: A comparison between female and male athletes and non-athletes.

This study compared the different factors associated with eating behaviors among young female and male athletes and non-athletes. A total of 580 female and male athletes and 362 female and male non-athletes between 10 and 19 years old participated. We used the subscales of the Eating Attitudes Test (EAT-26) to evaluate the factors associated with unhealthy eating behaviors. We found higher scores for females on the diet subscale compared with males, regardless of athletic group (P < 0.05). Non-athlete youths scored higher on this subscale compared with male athletes (P < 0.05). Our findings indicate higher scores for female athletes with regard to the bulimia and preoccupation with food subscale compared with other the groups (P < 0.05). Moreover, we observed that non-athlete males were more likely to engage in binge eating compared with athletes of the same sex (P < 0.05). Finally, females had higher scores on the oral self subscale than males, regardless of athletic group (P < 0.05). We concluded that the factors associated with eating behaviors differ with regard to sex and group.

Determinacion de la sensibilidad in vitro de diferentes cepas de Trypanosoma cruzi al benznidazol y al extracto de hoja de la planta Zanthoxylum chiloperone / Determination of the in vitro sensitivity of different Trypanosoma cruzi strains to benznidazole and the leaf extract of the plant Zanthoxylum chiloperone

Se evaluo la sensibilidad in vitro al benznidazol y al extracto de hojas de la planta Zanthoxylum chiloperone de diez cepas del Trypanosoma cruzi. Se utilizaron formas epimastigotas de las cepas, pertenecientes a diferentes linajes, diferente procedencia geografica, incluidas tres de Paraguay, y aisladas de hospederos distintos, abarcando humanos, triatominos y animales silvestres. El grado de sensibilidad a la droga y al extracto vegetal fue estimado por el porcentaje de lisis de los parasitos a las 24 y 48 horas de exposicion. Se observo un amplio rango de variacion en el porcentaje de lisis entre las cepas, de 22,2% a 90,8% a la concentracion del benznidazol de 200 ug/mL, con el hallazgo de una cepa con sensibilidad nula al mismo. Con respecto al extracto vegetal, tres concentraciones fueron testadas, de 500, 700 y 900 ug/mL, observandose variaciones en los porcentajes de lisis entre las cepas con las dos primeras concentraciones, entre un 2,1% y 100%, con lisis total en todas las cepas a la concentracion de 900 ug/mL. No se observo asociacion entre la diversidad de respuesta a los compuestos y la clasificacion de las cepas ni con su origen geografico y tipo de hospedero. Estas diferencias observadas resaltan la heterogeneidad de las poblaciones naturales del T. cruzi, aspecto importante a tener en cuenta en los estudios de sensibilidad a quimioterapeuticos y en los tamizajes primarios de nuevos antichagasicos. Asi mismo, se destaca la eficacia del extracto vegetal sobre diferentes cepas de este parasito.

A symbiotic gas exchange between bioreactors enhances microalgal biomass and lipid productivities: taking advantage of complementary nutritional modes.

This paper describes the association of two bioreactors: one photoautotrophic and the other heterotrophic, connected by the gas phase and allowing an exchange of O(2) and CO(2) gases between them, benefiting from a symbiotic effect. The association of two bioreactors was proposed with the aim of improving the microalgae oil productivity for biodiesel production. The outlet gas flow from the autotrophic (O(2) enriched) bioreactor was used as the inlet gas flow for the heterotrophic bioreactor. In parallel, the outlet gas flow from another heterotrophic (CO(2) enriched) bioreactor was used as the inlet gas flow for the autotrophic bioreactor. Aside from using the air supplied from the auto- and hetero-trophic bioreactors as controls, one mixotrophic bioreactor was also studied and used as a model, for its claimed advantage of CO(2) and organic carbon being simultaneously assimilated. The microalga Chlorella protothecoides was chosen as a model due to its ability to grow under different nutritional modes (auto, hetero, and mixotrophic), and its ability to attain a high biomass productivity and lipid content, suitable for biodiesel production. The comparison between heterotrophic, autotrophic, and mixotrophic Chlorella protothecoides growth for lipid production revealed that heterotrophic growth achieved the highest biomass productivity and lipid content (>22%), and furthermore showed that these lipids had the most suitable fatty acid profile in order to produce high quality biodiesel. Both associations showed a higher biomass productivity (10-20%), when comparing the two separately operated bioreactors (controls) which occurred on the fourth day. A more remarkable result would have been seen if in actuality the two bioreactors had been inter-connected in a closed loop. The biomass productivity gain would have been 30% and the lipid productivity gain would have been 100%, as seen by comparing the productivities of the symbiotic assemblage with the sum of the two bioreactors operating separately (controls). These results show an advantage of the symbiotic bioreactors association towards a cost-effective microalgal biodiesel production.

Parasitoides de dípteros coletados em excrementos de animais no sul de Goiás / Parasitoids of dipterous collected in animal excrements in Southern Goiás, Brazil

This study determined the species of microhymenopterous of Diptera present in cattle and buffalo dung, from May 2003 to June 2004 and from April 2006 to March 2007 in chicken manure. All samples were collected in Southern Goiás State, Brazil. The dipterous pupae were obtained by the flotation method. They were individually placed in gelatin capsules until the emergence of the flies and/or their parasitoids. The percentages of parasitism in cattle dung and chicken manure were 12.4% and 10.0%, respectively.

Blockade of presynaptic K ATP channels reduces the zinc-mediated posttetanic depression at hippocampal mossy fiber synapses.

Zinc is one of the most abundant transition metals in the brain, being present in a variety of synaptic processes. The mossy fiber terminals in area CA3 of the hippocampus contain large amounts of vesicular zinc and have an extremely high density of ATP-sensitive potassium (KATP) channels. The activation of these channels by zinc leads to rapid hyperpolarization of these presynaptic terminals and inhibition of transmitter release. It has been previously shown that intense stimulation of the synapses between mossy fibers and CA3 pyramidal cells evokes a posttetanic depression of synaptic activity, accompanied by a decrease in presynaptic calcium and vesicular zinc signals. These results suggest a neuromodulatory role for zinc at these synapses, which could be mediated by inhibition of presynaptic voltage-dependent calcium channels (VDCCs) and/or activation of presynaptic KATP channels. In order to evaluate the contribution of the second mechanism we have applied multiple tetanic stimulations in the absence and presence of the KATP channel blocker tolbutamide. Under control conditions, it was observed that the delivery of six tetani caused a posttetanic depression of synaptic activity. In the presence of tolbutamide, the depression was smaller and had a shorter time course. A similar depression was also observed in the presynaptic zinc and calcium signals. These findings are in agreement with the hypothesis that the activation of KATP channels by tetanically released zinc leads to cell hyperpolarization and subsequent reduction of presynaptic calcium entry, followed by the inhibition of both zinc and glutamate release. Thus, these results suggest that the inhibition of mossy fiber synaptic transmission by intensely released zinc is partially mediated by the activation of KATP channels.

Parasitóides de Diptera coletados em áreas florestal, rural e urbana em Monte Alegre, MG / Parasitoids of Diptera collected in forest, rural, and urban areas in Monte Alegre, MG

This study determined the species of parasitoids of Diptera present in forest, rural, and urban areas in the municipality of Monte Alegre, MG, from March to November 2006. The percentages of parasitism in forest, rural, and urban areas were 14.3 percent, 11.0 percent, and 18.8 percent, respectively. The most frequent species (34.7 percent) was Triplasta atrocoxalis (Ashmead) (Hymenoptera: Figitidae).

[Leg ulcer clinic at Santa Marta Hospital]. / Consulta de úlcera de perna do Hospital de Santa Marta.

Leg ulcers constitute a highly prevalent pathology in society, and are particularly common in the Angiology and Vascular Surgery outpatient clinic. The prevalence of these patients in this Department result from the fact that 70% of them display superficial and/or deep venous insufficiency of the lower limbs. To address this problem and optimize the therapeutic approaches available to the Chronic Leg Ulcer (CLU) patients, the Department of Angiology and Vascular Surgery has created, in March 2005, an appointment specific to CLU patients. An evaluation protocol was developed, including ulcer characterization, standardization of the conservative treatment, followed by surgical intervention, whenever required. The results obtained were evaluated 18 months after the onset of this protocol. Analysis of the results revealed that the majority of the patients responded positively to the new therapeutic approach, with closure of the ulcer in 43% of the patients and a significant improvement detected for an additional 30%. Furthermore, it was observed that a detailed evaluation of these patients should be regarded as a whole, followed by a standardized and targeted approach, resulting in a particularly successful approach on the treatment of this pathology.

Determinants of variable response to simvastatin treatment: the role of common variants of SCAP, SREBF-1a and SREBF-2 genes.

We investigated the effect of single-nucleotide polymorphisms in sterol regulatory element-binding factors-1a and -2 (SREBF-1a and SREBF-2) and SREBF cleavage-activating protein (SCAP) genes on lipid-lowering response to simvastatin. In all, 146 hypercholesterolemic patients of European descent were prospectively treated with simvastatin 20 mg/day for over 6 months. Of these 99 subjects completed the 6-month follow-up. Plasma lipids and lipoproteins were measured before and throughout the study. The mean percentage decrease in plasma total cholesterol (TC) was greater in subject carriers of SCAP 2386G allele compared with those homozygous for 2386A allele (-29.6+/-13.4 vs -22.1+/-13.8%, P=0.007). About 61% of the 2386G carriers were above-average responders for TC levels (DeltaTC -27.8%), whereas only 29% of 2386A homozygous reached this reduction (P=0.009). Our data suggest that the SCAP 2386A>G gene polymorphism was a significant predictor of TC and triglyceride responses to simvastatin treatment.

The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus.

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

Tetanically released zinc inhibits hippocampal mossy fiber calcium, zinc and synaptic responses.

At the zinc-enriched mossy fiber synapses from hippocampal CA3 area, electrical or chemical stimulation evokes zinc release from glutamatergic synaptic vesicles that may cause different pre- or postsynaptic actions. Besides zinc that can be co-localized with glutamate and GABA, the mossy fibers contain a very high density of ATP-sensitive potassium channels that are activated by zinc. We have investigated the possibility that intensely released zinc inhibits presynaptic calcium changes and consequently zinc and glutamate release. The studies were made combining optical recording of fast presynaptic calcium and zinc signals, using the fluorescent indicators Fura-2 and N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide, respectively, with measurements of field potentials. We have observed that strong tetanic stimulation caused posttetanic depressions of electrically induced presynaptic calcium and zinc signals and of synaptic responses, the depressions being blocked by zinc chelators. These results suggest that endogenously released zinc has an inhibitory role, mediated by presynaptic ATP-sensitive potassium channels and/or presynaptic calcium channels, that leads to the depression of zinc and glutamate release.

Acute ischemic hepatitis in aortocaval fistula.

OBJECTIVES: To characterise liver dysfunction in patients with aortocaval fistula. DESIGN: Retrospective study. MATERIALS: All four patients operated on for aortocaval fistula between 1999 and 2003. Three were males with ruptured abdominal aortic aneurysm (AAA). One was a female who underwent lumbar disk surgery. Four patients operated on for ruptured AAA were used as controls. METHODS: Measurement of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and creatinine from the preoperative period until full recovery. RESULTS: The median delay between the presumed formation of the fistula and surgery was 2 days (range 1-3 days). High levels of AST, ALT and LDH were observed in all patients, starting from the preoperative period, reaching the maximum on the first two postoperative days and normalising thereafter. Median peak values were: AST=4256 IU/l (range 816-7779), ALT=2487 IU/l (range 960-5645) and LDH=15165 IU/l (range 3122-32361). Serum creatinine also sustained alterations with a similar time course. Median peak values were: 256 micromol/l (range 230-468). All the patients survived. CONCLUSIONS: Acute ischemic hepatitis appears to be a consistent or, at least, a common complication of aortocaval fistula. Although a concern during the perioperative period, its course is benign and fully resolves upon successful surgery.

[Valvular transposition in patients with ulcers of the lower limbs following post thrombotic syndromes]. / A transposição valvular nos doentes com úlceras dos membros inferiores consequentes a síndromes pós-trombóticos.

Venous ulcers in patients with post thrombotic syndromes are complex situations with multiple therapeutic options. They are responsible for high morbidity rates, conservative treatment is very slow and recurrences are very common. Deep venous reconstructive surgery is an alternative, but it should be based on the morphologic and functional aspects of the venous system and only adopted after a very careful study, including venography. The authors describe a morphological "pattern", found in some of these patients and related to the competence of the saphenous femoral junction, rendering possible to perform a valvular transposition. Seven patients with post thrombotic ulcers who have been treated during the last 6 years in which the pattern already described was detect, underwent a transposition of the superficial femoral vein, to the great saphenous vein and when necessary complemented with skyn grafts. Before the operation all patients had ulcers with more than 3 cm in size (3.2-5.4 cm) and with more than 4 months duration (4-16 months). All ulcers healed in the postoperative period (mean time 28 days). All patients have been reevaluated in 2003 and it was diagnosed the thrombosis of two procedures, one of them with the recurrence of the ulcer who healed with conservative treatment. The authors consider this method as a very easy technique to perform, although rarely used, and a valid alternative in a highly selected group of patients.

Measurement of presynaptic zinc changes in hippocampal mossy fibers.

The hippocampal mossy fiber terminals of CA3 area contain high levels of vesicular zinc that is released in a calcium-dependent way, following high-frequency stimulation. However the properties of zinc release during normal synaptic transmission, paired-pulse facilitation and mossy fiber long-term potentiation are still unknown. Using the fluorescent zinc probe N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide, we measured fast mossy fiber zinc changes indicating that zinc is released following single and low levels of electrical stimulation. The observed presynaptic zinc signals are maintained during the expression of mossy fiber long-term potentiation, assumed to be mediated by an increase in transmitter release, and are enhanced during paired-pulse facilitation. This zinc enhancement is, like paired-pulse facilitation, reduced during established long-term potentiation. The correlation between the paired-pulse evoked zinc and field potential responses supports the idea that zinc is co-released with glutamate.