Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

Microscopia eletrônica de granulações em Proteus vulgaris tratado como Cloreto de Trifeniltetrazólio

Zonas citoplasmáticas de atividade redutora de TTC foram evidenciadas em células de Proteus vulgaris examinadas ao microscópio eletrõnico. As experiências foram realizadas em vários intervalos de tempo, sendo a reação, em alguns casos, estabilizada por meio de formol. Verificou-se que as granulações de formazana podiam ser removidas no interior do corpo bacteriano por meio de tratamento com acetona. O bombardeamento pelos eléctrons, quando os germes não tinham sido fixados com formol, determinou a saída das granulações do interior dos bacilos, deixando vestígios de sua presença (rompimento das membranas celulares nos pontos onde estavam localizados). A fixação com formol evitava a saída dos grânulos. O acúmulo dos cristais de formazana, internamente, nos pólos ou ao longo do corpo bacteriano, provoca sérias alterações morfológicas.

Diferenciação entre Listeria monocytogenes e Erysipelothrix rhusiopathiae com o clorêto de Trifeniltetrazólio

Experiências foram realizadas com bactérias dos gêneros Listeria e Erysipelothrix, em meios líquido e sólido, utilizando o clorêto de 2, 3, 5 - trifeniltetrazólio. A atividade enzimática redutora das listérias para o TTC foi diferente da do E. rhusiopathiae, principalmente em meio líquido e nas horas iniciais de observação. Preparações feitas para microscopia ótica e electrônica dos germes tratados com TTC revelaram a presença de granulações polares, bipolares e centrais dentro do corpo das listérias. A evidenciação de granulações coradas de formazana, intracelulares nas listérias, confirma estudos anteriores quanto à possibilidade da existência de mitocôndrias nas bactérias.

In vitro pollen germination and pollen viability in passion fruit (Passiflora spp.)

The use of Passiflora species for ornamental purposes has been recently developed, but little is known about pollen viability and the potential for crossing different species. The objective of this study was to evaluate the pollen viability of six Passiflora species collected from different physiological stages of development through in vitro germination and histochemical analysis using dyes. The pollen was collected in three stages (pre-anthesis, anthesis and post-anthesis). Three compositions of culture medium were used to evaluate the in vitro germination, and two dyes (2,3,5-triphenyltetrazolium chloride, or TTC, and Lugol's solution) were used for the histochemical analysis. The culture medium containing 0.03% Ca(NO3) 4H2O, 0.02% of Mg(SO4 ).7H2O, 0.01% of KNO3, 0,01% of H3BO3, 15% sucrose, and 0.8% agar, pH 7.0, showed a higher percentage of pollen grains germinated. Anthesis is the best time to collect pollen because it promotes high viability and germination. The Lugol's solution and TTC dye overestimated the viability of pollen, as all accessions showed high viability indices when compared with the results obtained in vitro.

Bidens mosaic virus: detecção via RT-PCR e identificação de Galinsoga parviflora como um novo hospedeiro natural do vírus

O Bidens mosaic virus (BiMV) é uma espécie tentativa do gênero Potyvirus, que infecta alface (Lactuca sativa). Na ausência de métodos eficientes para diagnose deste vírus, o objetivo do trabalho foi a síntese de oligonucleotídeos específicos e sua otimização em testes de RT-PCR em uma só etapa, partindo-se de extrações de RNA total. Os oligonucleotídeos 8851sens (5'AGG CAG TTC GCA CGG CAT AC 3´) e 9211ant (5´ CTT CAT CTG GAT GTG TGC TTC 3´) permitem a eficiente detecção do vírus e possibilitaram a descoberta de uma nova hospedeira do vírus, a planta Galinsoga parviflora, comumente encontrada em canteiros de produção comercial de alface.

Viabilidade polínica de Carica papaya L.: uma comparação metodológica

A viabilidade polínica do mamoeiro cultivar 'Sunrise Solo' (Carica papaya L.) foi estudada utilizando germinação in vitro e testes colorimétricos, assim como a validade dos testes colorimétricos como estimativa de viabilidade comparada àquela do teste germinativo. Os dois meios de cultura, descritos na literatura como meios eficientes para germinação da espécie, diferem basicamente pela presença de nutrientes essenciais e concentração de ágar. O meio de cultura sem elementos essenciais e com maior concentração de ágar forneceu o melhor índice de germinação polínica (65%). Os cinco corantes testados foram: 2,3,5-cloreto de trifeniltetrazólio (TTC), Alexander, carmim acético, lugol e Sudan IV. O teste de coloração com TTC forneceu estimativa de viabilidade (67,5%) equivalente ao teste de germinação in vitro e, portanto, confiável de viabilidade polínica. Os demais corantes testados superestimaram a viabilidade polínica (> 90%), porém são eficientes na determinação de constituintes celulares e da integridade do grão de pólen.

Validation of a simple and inexpensive method for the quantitation of infarct in the rat brain

A gravimetric method was evaluated as a simple, sensitive, reproducible, low-cost alternative to quantify the extent of brain infarct after occlusion of the medial cerebral artery in rats. In ether-anesthetized rats, the left medial cerebral artery was occluded for 1, 1.5 or 2 h by inserting a 4-0 nylon monofilament suture into the internal carotid artery. Twenty-four hours later, the brains were processed for histochemical triphenyltetrazolium chloride (TTC) staining and quantitation of the schemic infarct. In each TTC-stained brain section, the ischemic tissue was dissected with a scalpel and fixed in 10% formalin at 0ºC until its total mass could be estimated. The mass (mg) of the ischemic tissue was weighed on an analytical balance and compared to its volume (mm³), estimated either by plethysmometry using platinum electrodes or by computer-assisted image analysis. Infarct size as measured by the weighing method (mg), and reported as a percent (%) of the affected (left) hemisphere, correlated closely with volume (mm³, also reported as %) estimated by computerized image analysis (r = 0.88; P < 0.001; N = 10) or by plethysmography (r = 0.97-0.98; P < 0.0001; N = 41). This degree of correlation was maintained between different experimenters. The method was also sensitive for detecting the effect of different ischemia durations on infarct size (P < 0.005; N = 23), and the effect of drug treatments in reducing the extent of brain damage (P < 0.005; N = 24). The data suggest that, in addition to being simple and low cost, the weighing method is a reliable alternative for quantifying brain infarct in animal models of stroke.

Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications

Triphenyltetrazolium chloride (TTC) staining and echocardiography (ECHO) are methods used to determine experimental myocardial infarction (MI) size, whose practical applicability should be expanded. Our objectives were to analyze the accuracy of ECHO in determining infarction size in rats during the first days following coronary occlusion and to test whether a simplified single measurement by TTC correctly indicates MI size, as determined by the average value for multiple slices. Infarction was induced in female Wistar rats by coronary artery occlusion and MI size analysis was performed after the acute (7th day) and chronic periods (after 4 weeks) by ECHO matched with TTC. ECHO and TTC showed similar values of MI size (% of left ventricle perimeter) in acute (ECHO: 33 ± 11, TTC: 35 ± 14) and chronic (ECHO: 38 ± 14, TTC: 39 ± 13 periods), and also presented an excellent correlation (r = 0.92, P < 0.001). Although measurements from different heart planes showed discrepancies, a single measurement acquired from the mid-ventricular level by TTC was a good estimate of MI size calculated by the average of multiple planes, with minimal disagreement (Bland-Altman test with mean ratio bias of 0.99 ± 0.07) and close to an ideal correlation (r = 0.99, P < 0.001). In the present study, ECHO was confirmed as a useful method for the determination of MI size even in the acute phase. Also, the single measure of a mid-ventricular section proposed as a simplification of the TTC method is a satisfactory prediction of average MI extension.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

Comparação entre os métodos rápidos SimplateR TPC- CI e PetrifilmR AC e os métodos convencionais de contagem em placas para a enumeração de aeróbios mesófilos em sorvetes

A contagem de aeróbios mesófilos ou contagem padrão em placas em um produto alimentício reflete a qualidade da matéria-prima, bem como as condições de processamento, manuseio e estocagem. O método de contagem em placas tradicionais requer 48h de incubação para a posterior leitura dos resultados. Comparou-se dois métodos que parecem ser uma boa alternativa para a contagem padrão em placas: SimplateR TPC-CI e PetrifilmR AC. Também avaliou-se a capacidade inibitória do 2, 3, 5 cloreto de trifeniltetrazólio quando adicionado ao Agar Plate Count. Este corante é incolor na forma oxidada e vermelho quando reduzido pelos microrganismos, devido à formação de formazano. O coe-ficiente de correlação obtido, através de estudos conduzidos com 60 amostras de sorvetes, entre 0,866-0,979 indicou uma equivalente sensibilidade dos métodos testados. Somente a contagem padrão em placas com TTC diferiu signicativamente da contagem padrão em placas.

Tetrazolium test for evaluating triticale seed viability

Freshly harvested triticale seeds are usually dormant, making the immediate evaluation of the physiological potential of seed lots difficult. We evaluated different triphenyl tetrazolium chloride (TTC) test methods for rapidly determining the viability of four seed lots of x.Triticosecale Wittmack cultivar IPR111. The test variables were: Preconditioning, (i) placing whole seeds between moistened paper towels or (ii) directly soaking the seeds in water, both procedures being conducted at 20 ºC for 18 hours; Post-conditioning seed preparation, (i) longitudinal bisection of the seed through the embryo with one half being stained and the other discarded or (ii) longitudinal bisection with both halves being stained; Staining for three and four hours, in the dark, with 0.1%, 0.5% or 1.0% (w/v) TTC according to the preconditioning method described above, (i) both halves of each seed were placed on filter paper moistened with TTC and maintained at 40 ºC or (ii) one half of each seed was immersed in 5 mL of TTC solution in a 100 mL glass beaker at 30 ºC. The best results were obtained by preconditioning seeds between moistened paper towels at 20 ºC for 18 hours and staining on filter paper with 1.0% (w/v) TTC for three hours at 40 ºC.