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.

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.

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.

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.

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.

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.

[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.

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.

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.

Hippocampal mossy fiber calcium transients are maintained during long-term potentiation and are inhibited by endogenous zinc.

The hippocampal mossy fiber long-term potentiation (LTP) is an N-methyl-d-aspartate (NMDA) receptor-independent form of long-lasting synaptic plasticity characteristic of the zinc-enriched mossy fiber synapses. Its expression is generally considered to have a presynaptic locus and to be mediated by a persistent increase of evoked transmitter release. Because the release process is calcium-dependent, the observed increase in synaptic efficacy could be due to a persistent modification of presynaptic calcium mechanisms, triggered by the large calcium influx associated with long-term potentiation induction. Alternatively, it might be caused by an enhancement in the sensitivity to calcium of some components of the synaptic vesicle release system, following the large intraterminal calcium accumulation. We investigated the first hypothesis by measuring presynaptic Fura-2 calcium signals associated with electrically induced mossy fiber long-term potentiation. We have observed that like residual calcium, single presynaptic calcium changes are not enhanced during the maintenance phase of mossy fiber long-term potentiation. This result supports the idea that this form of long-term potentiation may be mediated by persistent changes of some process occurring after calcium entry. It has been established that voltage-dependent calcium channels are inhibited by zinc and that endogenous zinc is released in a calcium-dependent way following intense mossy fiber activation. Because there is evidence that at these synapses zinc is also released following single electrical stimulation, we investigated the effect of endogenous zinc on single presynaptic calcium signals and on field potentials associated with mossy fiber LTP. We have observed that this form of LTP could be induced in the presence of the permeant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and that application of this chelator, during LTP, caused an enhancement of the presynaptic calcium signals without affecting synaptic transmission. This enhancement is consistent with the idea that mossy fiber zinc, released following individual stimuli, inhibits presynaptic calcium mechanisms.

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.

The effect of bisbenzylisoquinoline alkaloids on Trypanosoma cruzi infections in mice.

Five bisbenzylisoquinoline (BBI) alkaloids, curine, cycleanine, isotet:andrine, limacine and pheanthine were tested for trypanocidal activity in C 3H He mice infected with Y or CL strain of Trypanosoma cruzi. The activity was compared with the baseline drug, benznidazole. Oral treatment was more effective with curine at 10 mg/kg or with cycleanine at 2 mg/kg daily for 10 days in mice infected with Y or CL strain. In these groups, the parasitemias were negative after 5-7 weeks after inoculation and mortality time 50 (MT(50)) was significantly higher than untreated mice. Benznidazole was effective in mice infected with CL strain but not in mice infected with Y strain. The other BBI showed a relative efficacy against both strains. The effect of BBI alkaloids could be due to a blocking of the Ca2+ channel for the regulation of T. cruzi infectivity to invade host cells or their selective immunosuppressive properties.

Efficacy of the bisbenzylisoquinoline alkaloids in acute and chronic Trypanosoma cruzi murine model.

We have shown previously that daphnoline and cepharanthine are active against Trypanosoma cruzi and inhibited trypanothione reductase. The effects of oral treatments with daphnoline, cepharanthine and benznidazole were examined in Balb/c mice infected with T. cruzi acutely and chronically. In acute infections, parasitaemia was significantly reduced in the daphnoline-treated mice compared with controls and benznidazole-treated mice. The parasitological cure rate was increased in mice treated with daphnoline. Fifty days after infection, the negative serological response in both models was significantly different for the three tested drugs. Daphnoline showed the highest negative serological rate (48%). In chronically infected mice treated with daphnoline, we were unable to detect parasites in 70% of mice. The results obtained of oral treatment of daphnoline suggest that this bisbenzylisoquinoline may be useful in the treatment of acute and chronic Chagas' disease. This was not seen with cepharanthine, an excellent trypanothione reductase inhibitor.

Cryptofolione derivatives from Cryptocarya alba fruits.

Cryptofolione (1) and the new cryptofolione derivative 6-(4,6-dimethoxy-8-phenyl-octa-1,7-dienyl)-4-hydroxy-tetrahydro-pyran-2-one (2) were isolated from the fruits of Cryptocarya alba. The structures were elucidated by spectroscopic methods. Cryptofolione showed activity towards Trypanosoma cruzi trypomastigotes, reducing their number by 77% at 250 microg mL(-1). Cryptofolione showed moderate cytotoxicity in both macrophages and T. cruzi amastigotes. It also displayed a mild inhibitory effect on the promastigote form of Leishmania spp. As both cytotoxic and trypanocidal effects are similar, the compound presented little selectivity in our assay models.

Brazilian population database for the 13 STR loci of the AmpFlSTR Profiler Plus and Cofiler multiplex kits.

Allele frequencies for the 13 STR core loci (D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, CSF1PO, TPOX, THO1 and D16S539) included in the AmpFlSTR((R)) Profiler Plus and AmpFlSTR((R)) Cofiler kits were obtained for a sample of 700-800 genetically unrelated Brazilians. The expected performance of these loci for personal identification and paternity testing in the Brazilian population was estimated.

Activity of compounds isolated from Chilean lichens against experimental cutaneous leishmaniasis.

Three secondary metabolites isolated from Chilean lichens, (+) usnic acid, pannarine and 1'-chloropannarine, were tested against promastigotes forms of three strains of Leishmania ssp. Pannarine and 1'-chloropannarine exhibited in vitro activity at 50 micrograms/ml and (+) usnic acid at 25 micrograms/ml. BALB/c mice infected with Leishmania amazonensis were treated 4 weeks post-infection with (+) usnic acid by subcutaneous or oral routes for 15 days at 25 mg/kg or by five intralesional injections at interval of 4 days at 25 mg/kg of body weight. The reference drug, N-methylglucamine antimonate (Glucantime), was administered by subcutaneous injections (regimens of 28 mg of pentavalent antimony) for 15 days. The subcutaneous and oral treatments with (+) usnic did not produce any effect, but by intralesional administration we observed a significant effect that reduced by 43.34% the weight lesions and by 72.28% the parasites loads in infected footpads.

Leishmanicidal activity of two canthin-6-one alkaloids, two major constituents of Zanthoxylum chiloperone var. angustifolium.

The crude alkaloidal extract of Zanthoxylum chiloperone stem bark exhibited in vitro activity against various strains of Leishmania ssp. at 100 microg/ml. Two active major constituents were isolated and identified as canthin-6-one and 5-methoxycanthin-6-one. The effect of these compounds was also tested in an in vivo assay using BALB/c mice infected with Leishmania amazonensis. The mice were treated for 5 weeks postinfection with these alkaloids by oral (14 days) or intralesional route (4 days) at 10 mg/kg daily. The reference drug, N-methylglucamine antimonate was administered by subcutaneous injections at 100 mg/kg for 10 days. Intralesional administration of canthin-6-one reduced the parasite burden but not significantly when it was compared with the untreated group, while the reference drug reduced by 91% the parasite loads in the lesion.

[Phlebotomine behavior in forest and extraforest environments, in an endemic area of American cutaneous leishmaniasis in northern Paraná State, southern Brazil]. / Observações sobre o comportamento de flebotomíneos em ecótopos florestais e extraflorestais, em área endêmica de leishmaniose tegumentar americana, no norte do Estado do Paraná, sul do Brasil.

Between November 1988 and April 1990, 75,637 phlebotomines were captured with the use of Falcão light traps on Palmital farm in the north of Paraná State, Brazil. Fifteen species were represented. The percentage of 95.8% of the insects were captured within the domiciliary area and the remaining 4.2% in the forest environment. The percentage of 62 were captured in one single Falcão light trap installed in a henhouse, where Blancasmya migonei predominated. In all the other traps Psychodopygus whitmani was predominant. B. migonei, P. whitmani, Pintomyia pessoai, Pintomyia fischeri and Psychodopygus intermedius are highly significant vectors of American cutaneous leishmaniasis and represented 93.8% of all phlebotomines captured with 10 Falcão light traps. The monthly densities of these insects were obtained from one Falcão light trap installed in a henhouse and increased especially in the warmer and more humid months. In the forest environment the phlebotomines were captured principally in traps installed about 10 meters above the ground and P. whitmani, B. migonei, P. intermedius, Brumptomyia brumpti, Psychodopygus monticola and P. pessoai were the dominant species. The need for information about the epidemiology of American cutaneous leishmaniasis calls for further investigation with a view to clarifying the implications of the relationship between the phlebotomines and domestic animals in the Leishmania cycle of transmission in domiciliary areas.

[American cutaneous leishmaniasis: phlebotominae of the area of transmission in the north of Paraná, Brazil]. / Leishmaniose tegumentar americana: flebotomíneos de área de transmissão no Norte do Paraná, Brasil.

Most of the cases of cutaneous leishmaniasis diagnosed in the laboratory of the State University of Maringá, Paraná, Brazil, are related to areas of northern Paraná State, which have been settled since 40 years age, when the destructions of vast areas of forest in this region was begun. The native vegetation, already, in large part, destroyed is constituted of dense forest of the type found in the transition from tropical to subtropical regions. The landscape is constituted of plateau with a climate of transition from tropical to subtropical and annual mean temperatures of 20 degrees C to 22 degrees C, the mean of the hottest month being superior to 22 degrees C. The native forests are represented by small, sparse patches of vegetation, with some modification in the basins of the rivers Ivaí, Paranapanema and Paraná and their tributaries. In this environment 24 captures of phlebotomines were made--2 per month from October 1986 to September 1987, between 6 P.M. and 1 A.M. The captures with Shannon trap were made in the outlying forest of Palmital farm. The majority of the 16,496 phlebotomines were classified into 13 species. Lutzomyia whitmani was predominant with 11,188 (67.82%) specimens, followed by Lutzomyia intermedia with 2,900 (17.58%) and Lutzomyia migonei with 1,481 (9.03%). Lutzomyia whitmani and Lutzomyia intermedia have been manifesting great capacity of adaptation in modified environments, where they have played a significant role in transmission of leishmaniasis.

[Adult culicidae captured in the southern region of Brazil]. / Nota sobre culicídeos adultos coletados na região sul do Brasil.

During the period from September 1988 to April 1990, mosquitoes were captured using Shannon light trap and Falcão light traps, in Terra Boa county, in the north of Paraná State, Brazil. The Shannon trap was installed in the outlying modified forest and Falcão traps were installed in the modified forest and domiciliary areas. The prevalence of species was verified and the methods of capture compared.