Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis.

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the "black box", remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding.

PPARs are members of the nuclear hormone receptor superfamily and are primarily involved in lipid metabolism. The expression patterns of all 3 PPAR isotypes in 22 adult rat organs were analyzed by a quantitative ribonuclease protection assay. The data obtained allowed comparison of the expression of each isotype to the others and provided new insight into the less studied PPAR beta (NR1C2) expression and function. This isotype shows a ubiquitous expression pattern and is the most abundant of the three PPARs in all analyzed tissues except adipose tissue. Its expression is especially high in the digestive tract, in addition to kidney, heart, diaphragm, and esophagus. After an overnight fast, PPAR beta mRNA levels are dramatically down-regulated in liver and kidney by up to 80% and are rapidly restored to control levels upon refeeding. This tight nutritional regulation is independent of the circulating glucocorticoid levels and the presence of PPAR alpha, whose activity is markedly up-regulated in the liver and small intestine during fasting. Finally, PPAR gamma 2 mRNA levels are decreased by 50% during fasting in both white and brown adipose tissue. In conclusion, fasting can strongly influence PPAR expression, but in only a few selected tissues.

Residues of b-lactams and quinolones in tissues and milk samples. Confirmatory analysis by liquid chromatography-mass spectrometry

The aim of this work is to optimize and validate methods for the multiresidue determination of series of families of antibiotics as quinolones, penicillins and cephalosporins included in European regulation in food samples using LC-MS/MS. Different extraction techniques and clean-up applied to antibiotics in meat were compared. The quality parameters were established according with EU guideline. The developed method was applied to 49 positive raw milk samples from animal medicated with different antibiotics; the 63% of the analyzed samples were found to be compliant. ___________________________________________________________________________________________

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Analysis of biological tissues in infant chest for the development of an equivalent radiographic phantom

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

A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes

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

Chemical analysis of different larval instars and tissues of Pachycondyla (=Neoponera) villosa (Hymenoptera: Formicidae, Ponerinae)

Chemical analyses of complete larvae of the first to third instar and cuticle, fat body and salivary glands extracts of fourth instar larvae using gas chromatography and gas chromatography-mass spectrometry, were performed upon Pachycondyla villosa. The results revealed that P. villosa larvae do not produce a pheromone, as only fatty acids and n-alkanes were detected. After quantifying the identified compounds, it was determined that the fat body is the main place of storage and/or production of the cuticular hydrocarbons. It was also observed that the absolute quantity of cuticular hydrocarbons increases progressively during larval development. Inferences about the transport behavior of matured larvae to the pupation place and the colony odor are discussed.

Microwave fixation in rat fetuses tissues: Histological and immunohistochemical analysis

The aim of this study was to show the microwaves action in fixation of rat fetuses, dermal and cartilaginous tissues, using histological and immunohistochemistry methods for analysis. It was possible to conclude in this study using the rat as experimental model that the two methods for antibody retrieval, presented an excellent ways for the use of Ki67 antibody in the immunohistochemical analysis.

Matrix effect on butyltin analysis of sediments and fish tissues by GC-PFPD

Butyltin (BTs) quantification in environmental matrices can be affected by interfering species found primarily in complex matrices,such as sediment and biota tissues. This study investigated matrix effects in analytical procedures for butyltin (TBT,DBT and MBT) quantification and speciation in sediments and in two fish tissues (gill and liver) by gas chromatography with pulsed flame photometric detection (GC-PFPD) analysis. Unlike sediment samples,tissues exhibited a significant matrix effect,thus,the quantification should be made by curve over matrix to avoid quantification errors and loss of analytical accuracy. © 2013 Sociedade Brasileira de Química.

Traceability of poultry offal meal in broiler feeding using isotopic analysis (delta C-13 and delta N-15) of different tissues

Our goal was to trace the inclusion of poultry offal meal (OM) in diets by using carbon (13C/12C) and nitrogen (15N/14N) isotopic ratios of different tissues in order to contribute for the development of an independent technology for the certification of the feeding of broilers reared on diets with no addition of animal ingredients. Eighty one-day-old chicks were randomly distributed into five experimental treatments, that is, diets containing increasing levels of OM inclusion (0, 2, 4, 8 and 16% OM), with four replicates of four birds each. At 42 days of age, four birds per treatment (n=4) were randomly selected, weighed, and sacrificed to collect breast muscle (Pectoralis major), keel and tibia samples to determine their isotopic ratios (13C/12C e 15N/14N). It was observed that 13C and 15N enrichment increased as a function of increasing OM inclusion in all diets. The analyses of the Pectoralis major showed that that only treatments with 8 and 16% OM dietary inclusion were different form those in the control group (0% OM). on the other hand, when the keel and tibia were analyzed, in addition to 8 and 16% OM), the treatment with 4% OM inclusion was also different from the control group. The use of isotopic ratios of stable carbon and nitrogen isotopes is an alternative to trace OM inclusion in broiler diets as it is capable of tracing OM levels below those usually practiced by the poultry industry in Brazil.

Histological analysis of effects of 24% EDTA gel for nonsurgical treatment of periodontal tissues

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Analysis of the peri-implant soft tissues in contact with zirconia abutments: an evidence-based literature review

The aim of this study is to evaluate through a literature review, the soft tissue response in contact with zirconia abutments, including case reports comparing prosthetics rehabilitations with zirconia and titanium abutments upto 3 years of follow-up as well as the factors that should be considered on implant's abutment selection. Metallic abutments can provide grayish color when in contact with thin soft tissues which may lead the implant prosthetic treatment to failure. In this context, the abutments of zirconia stand out because there is an excellent linking between esthetics and the health of peri-implant soft tissues. A consult of the published researches was made on the PubMed database from 2000 to September 2012. The including criteria were: literature reviews, clinical studies and case reports in English that focused on the response of the soft tissue in contact with zirconia implant abutments. The studies that were not in English and did not match the tackled issue were excluded. A total of 32 articles were found. According to the search strategy, just 16 articles were selected for this review. Three studies affirmed that zirconia abutments have an excellent soft tissue response; one study showed increased gingival recession with zirconia abutments and nine studies do not stand out any difference on biological behavior between titanium and zirconia abutments. Three studies affirmed that zirconia abutments provide natural gingival appearance, anatomic contour and greater esthetics. The use of zirconia abutments is recommended for anterior regions because of their greater optical properties and esthetic results and more studies should be performed and analyzed longitudinally regarding their biological response. The zirconia abutments have been established to be essential in order to achieve great esthetic results in cases of thin peri-implant soft tissues and in regions where the three-dimensional placement of implants is more superficial.

Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin

Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription-polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow-derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue-derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.

A comprehensive test system to identify suitable antibodies against p53 for immunohistochemical analysis of canine tissues

The tumour suppressor p53 is commonly detected in tissues of companion animals by means of antibodies raised against the human protein. The following three-step procedure was devised to test the suitability of such antibodies for immunohistochemistry on canine tissues. (1) Western blot and immunohistochemical analyses on bacterially expressed recombinant canine protein to assess human-to-canine cross-reactivity. (2) Immunohistochemistry of cultured, UVB-irradiated canine keratinocytes to evaluate suitability for detection of endogenous p53. (3) Immunohistochemistry on tissue arrays to further substantiate suitability of the antibodies on a panel of normal and neoplastic human and canine tissues. Five of six antibodies cross-reacted with recombinant canine p53. Three of these (PAb122, PAb240, CM-1) also immunolabelled stabilized wild type p53 in cell cultures and elicited a consistent, characteristic labelling pattern in a subset of tumours. However, two alternative batches of polyclonal antibody CM-1 failed to detect p53 in cell cultures, while showing a characteristic labelling pattern of a completely different subset of tumours and unspecific labelling of normal tissues. The test system described is well suited to the selection of antibodies for immunohistochemical p53 detection. The results emphasize the need to include appropriate controls, especially for polyclonal antibodies.