Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands (1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and 300 bp) expressed specifically in the cultivated bone marrow cell group. Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification of small differences in gene expression of bone marrow cells in two defined conditions. Thus, we expect that DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in several stem cell types under distinct conditions.

Determination of 2-methylimidazole and 4-methylimidazole in caramel colors by capillary electrophoresis

The use of chemical preservative compounds is common in the food products industry. Caramel color is the most usual additive used in beverages, desserts, and breads worldwide. During its fabrication process, 2- and 4-methylimidazole (MeI), highly carcinogenic compounds, are generated. In these cases, the development of reliable analytical methods for the monitoring of undesirable compounds is necessary. The primary procedure for the analysis of 2- and 4-MeI is using LC- or GC-MS techniques. These procedures are time-consuming and require large amounts of organic solvents and several pretreatment steps. This prevents the routine use of this procedure. This paper describes a rapid, efficient, and simple method using capillary electrophoresis (CE) for the separation and determination of 2- and 4-MeI in caramel colors. The analyses were performed using a 75 μm i.d. uncoated fused-silica capillary with an effective length of 40 cm and a running electrolyte consisting of 160 mmol L-1 phosphate plus 30% acetonitrile. The pH was adjusted to 2.5 with triethylamine. The analytes were separated within 6 min at a voltage of 20 kV. Method validation revealed good repeatability of both migration time (<0.8% RSD) and peak area (<2% RSD). Analytical curves for 2- and 4-MeI were linear in the 0.4-40 mg L-1 concentration interval. Detection limits were 0.16 mg L-1 for 4-MeI and 0.22 mg L-1 for 2-MeI. The extraction recoveries were satisfactory. The developed method showed many advantages when compared to the previously used method. © 2013 American Chemical Society.

Determination of sildenafil and vardenafil by capillary zone electrophoresis using capacitively coupled contactless conductivity detection

A method based on capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) for determination of two important phosphodiesterase type-5 inhibitors (sildenafil and vardenafil) is introduced. The background electrolyte (BGE) consisted of an aqueous solution of 500 mmol L-1 acetic acid, and the capillary was previously treated with polybrene solution to prevent cationic analytes from adsorbing onto the inner surface. Although the analytes migrate in the counter flow, the total time is short. An instrument with two C4D detectors allowed a seamless transition from a fast method (less than one minute) but of low-efficiency using the first detector to a more efficient method using the second detector. The analysis of commercial tablets showed no significant difference between CE-C4D and HPLC methods. Conductivity detection is a well-known low selectivity detection scheme, which in conjunction with the high mobility of the co-ion in the BGE (hydroxonium) allows one to predict that other cationic analogues of sildenafil can also be detected. This is an interesting feature given the increasing number of compounds in this class. © 2013 The Royal Society of Chemistry.

Caracterização molecular parcial e localização subcelular de um isolado brasileiro do vírus do mosaico do feijoeiro do sul dos E.U.A

Pós-graduação em Genética - IBILCE

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

O Estado do Pará é o principal produtor brasileiro de pimenta-do-reino (Piper nigrum Link), entretanto a sua produção tem sido bastante afetada pela doença conhecida como fusariose. O Fusarium solani f. sp. piperis é o agente causador desta doença que afeta o sistema radicular da planta, causando o apodrecimento das raízes e a queda das folhas levando à morte da planta. Algumas piperáceas nativas da região amazônica, entre elas a espécie Piper tuberculatum Jacq., têm se mostrado resistentes à infecção pelo F. solani f. sp. piperis, e desta forma têm sido utilizadas em estudos de interação planta-patógeno. Neste trabalho foram avaliadas cinco condições de extração de proteínas com o objetivo de selecionar tampões adequados para a extração de proteínas totais de folhas e raízes de P. tuberculatum. Os tampões utilizados para a extração de proteínas de raízes e folhas foram: tampão salino, tampão sacarose, tampão glicerol, tampão uréia e tampão fosfato de sódio. As análises quantitativas mostraram que os tampões sacarose, glicerol e uréia foram mais eficientes na extração de proteínas de folhas e raízes. Análises de SDS-PAGE mostraram padrões diferenciados de bandas em extratos protéicos de folhas e raízes obtidos com os diferentes tampões. Os resultados obtidos neste trabalho contribuem para a identificação de tampões de extração adequados para a obtenção de amostras de proteínas totais em estudos de interação P. tuberculatum - F. solani f. sp. piperis.

Selênio em tilápia do Nilo utilizando eletroforese em gel e espectrometria atômica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of whitening gel on pulp chamber temperature rise by in-office bleaching technique

INTRODUÇÃO: O clareamento dental representa uma manobra conservadora na recuperação estética de dentes com alterações cromáticas. Contudo, os tratamentos clareadores são passíveis de causar efeitos adversos, quando não bem planejados e executados. OBJETIVO: Este estudo avaliou a influência do gel clareador no aumento da temperatura intra-câmara pulpar através da técnica de clareamento dental fotoativado realizado em consultório. MATERIAL E MÉTODO: Um incisivo central superior humano foi seccionado na porção radicular 3 mm abaixo da junção cemento-esmalte. O canal radicular foi alargado para permitir a introdução do sensor do termômetro na câmara pulpar, a qual foi preenchida com pasta térmica, favorecendo a transferência de calor das paredes dentárias para o sensor do termômetro digital com termopar tipo K (MT- 401A) durante o clareamento. Três agentes clareadores fotossensíveis (peróxido de hidrogênio a 35%) foram utilizados, sendo: Whiteness HP (FGM), Whiteness HP Maxx (FGM) e Lase Peroxide Sensy (DMC). Um aparelho fotopolimerizador de led (Flash Lite - Discus Dental) foi empregado para a ativação dos géis clareadores. Seis ciclos de clareamento foram realizados para cada grupo testado. Os resultados foram submetidos à ANOVA de um critério e ao teste t (LSD) (α<0,05). RESULTADO: A menor média de variação de temperatura (ºC) foi observada com o Lase Peroxide Sensy (0,20), enquanto que a maior com o Whiteness HP (1,50). CONCLUSÃO: Os géis clareadores Whiteness HP e Whiteness HP Maxx interferiram significativamente no aumento da temperatura intra-câmara pulpar durante o clareamento, sendo esta variação dependente do tipo de gel clareador empregado.

Perfil eletroforético, características estruturais e fertilidade in vitro do sêmen de touros Nelores submetidos à degeneração testicular por insulação

Pós-graduação em Medicina Veterinária - FMVZ

Preparation of TiO2/SnO2 Thin Films by Sol-Gel Method and Periodic B3LYP Simulations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mineral Loss and Morphological Changes in Dental Enamel Induced by a 16% Carbamide Peroxide Bleaching Gel

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of cryoprotectants on the reconstitution of silica nanoparticles produced by sol-gel technology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Improved electrical transport in lightly Er-doped sol-gel spin-coating SnO2 thin films, processed by photolithography

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Rietveld refinement and optical properties of SrWO4:Eu3+ powders prepared by the non-hydrolytic sol-gel method

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Functional and structural studies of the disulfide isomerase DsbC from the plant pathogen Xylella fastidiosa reveals a redox-dependent oligomeric modulation in vitro

Xylella fastidiosa is a Gram-negative bacterium that grows as a biofilm inside the xylem vessels of susceptible plants and causes several economically relevant crop diseases. In the present study, we report the functional and low-resolution structural characterization of the X. fastidiosa disulfide isomerase DsbC (XfDsbC). DsbC is part of the disulfide bond reduction/isomerization pathway in the bacterial periplasm and plays an important role in oxidative protein folding. In the present study, we demonstrate the presence of XfDsbC during different stages of X. fastidiosa biofilm development. XfDsbC was not detected during X. fastidiosa planktonic growth; however, after administering a sublethal copper shock, we observed an overexpression of XfDsbC that also occurred during planktonic growth. These results suggest that X. fastidiosa can use XfDsbC in vivo under oxidative stress conditions similar to those induced by copper. In addition, using dynamic light scattering and small-angle X-ray scattering, we observed that the oligomeric state of XfDsbC in vitro may be dependent on the redox environment. Under reducing conditions, XfDsbC is present as a dimer, whereas a putative tetrameric form was observed under nonreducing conditions. Taken together, our findings demonstrate the overexpression of XfDsbC during biofilm formation and provide the first structural model of a bacterial disulfide isomerase in solution. Structured digital abstract XfDsbC and XfDsbC bind by x ray scattering (View Interaction: 1, 2) XfDsbC and XfDsbC bind by molecular sieving (View interaction) XfDsbC and XfDsbC bind by comigration in non denaturing gel electrophoresis (View interaction) XfDsbC and XfDsbC bind by cross-linking study (View Interaction: 1, 2) XfDsbC and XfDsbC bind by dynamic light scattering (View Interaction: 1, 2)