Instantaneous characterization of vegetable oils via TAG and FFA profiles by easy ambient sonic-spray ionization mass spectrometry

A fast and reliable method is presented for the analysis of vegetable oils. Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) is shown to efficiently desorb and ionize the main oil constituents from an inert surface under ambient conditions and to provide comprehensive triacylglyceride (TAG) and free fatty acid (FFA) profiles detected mainly as either [ TAG + Na](+) or [FFA - H](-) ions. EASI(+/-)-MS analysis is simple, easily implemented, requires just a tiny droplet of the oil and is performed without any pre-separation or chemical manipulation. It also causes no fragmentation of TAG ions hence diacylglyceride (DAG) and monoacylglyceride (MAG) profiles and contents can also be measured. The EASI(+/-)-MS profiles of TAG and FFA permit authentication and quality control and can be used, for instance, to access levels of adulteration, acidity, oxidation or hydrolysis of vegetable oils in general.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Hepatic denervation impairs the assembly and secretion of VLDL-TAG

VLDL secretion is a regulated process that depends on the availability of lipids, apoB and MTP. Our aim was to investigate the effect of liver denervation upon the secretion of VLDL and the expression of proteins involved in this process. Denervation was achieved by applying a 85% phenol solution onto the portal tract, while control animals were treated with 9% NaCl. VLDL secretion was evaluated by the Tyloxapol method. The hepatic concentration of TAG and cholesterol, and the plasma concentration of TAG, cholesterol, VLDL-TAG, VLDL-cholesterol and HDL-cholesterol were measured, as well as mRNA expression of proteins involved in the process of VLDL assembly. Hepatic acinar distribution of MTP and apoB was evaluated by immunohistochemistry. Denervation increased plasma concentration of cholesterol (125.3 +/- 10.1 vs. 67.1 +/- 4.9 mg dL(-1)) and VLDL-cholesterol (61.6 +/- 5.6 vs. 29.4 +/- 3.3 mg dL(-1)), but HDL-cholesterol was unchanged (45.5 +/- 6.1 vs. 36.9 +/- 3.9 mg dL(-1)). Secretion of VLDL-TAG (47.5 +/- 23.8 vs. 148.5 +/- 27.4 mg dL h(-1)) and mRNA expression of CPT I and apoB were reduced (p < 0.01) in the denervated animals. MTP and apoB acinar distribution was not altered in the denervated animals, but the intensity of the reaction was reduced in relation to controls. Copyright (C) 2008 John Wiley & Sons, Ltd.

Amazonian Vegetable Oils and Fats: Fast Typification and Quality Control via Triacylglycerol (TAG) Profiles from Dry Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry Fingerprinting

Amazonian oils and fats display unique triacylglycerol (TAG) profiles and, because of their economic importance as renewable raw materials and use by the cosmetic and food industries, are often subject to adulteration and forgery. Representative samples of these oils (andiroba, Brazil nut, buriti, and passion fruit) and fats (cupuacu, murumuru, and ucuba) were characterized without pre-separation or derivatization via dry (solvent-free) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Characteristic profiles of TAG were obtained for each oil and tat. Dry MALDI-TOF MS provides typification and direct and detailed information, via TAG profiles, of their variable combinations of fatty acids. A database from spectra could be developed and may be used for their fast and reliable typification, application screening, and quality control.

Dispersal and survival of Nyssomyia intermedia and Nyssomyia neivai (Diptera: Psychodidae: Phlebotominae) in a cutaneous leishmaniasis endemic area of the speleological province of the Ribeira Valley, state of São Paulo, Brazil

The dispersal and survival of the phlebotomines Nyssomyia intermedia and Nyssomyia neivai (both implicated as vectors of the cutaneous leishmaniasis agent) in an endemic area was investigated using a capture-mark-release technique in five experiments from August-December 2003 in municipality of Iporanga, state of São Paulo, Brazil. A total of 1,749 males and 1,262 females of Ny. intermedia and 915 males and 411 females of Ny. neivai were marked and released during the five experiments. Recapture attempts were made using automatic light traps, aspiration in natural resting places and domestic animal shelters and Shannon traps. A total of 153 specimens (3.48%) were recaptured: 2.59% (78/3,011) for Ny. intermedia and 5.35% (71/1,326) for Ny. neivai. Both species were recaptured up to 144 h post-release, with the larger part of them recaptured within 48 h. The median dispersion distances for Ny. intermedia and Ny. neivai, respectively, were 109 m and 100 m. The greatest dispersal range of Ny. intermedia was 180 m, while for Ny. neivai one female was recaptured in a pasture at 250 m and another in a pigsty at 520 m, showing a tendency to disperse to more open areas. The daily survival rates calculated based on regressions of the numbers of marked insects recaptured on the six successive days after release were 0.746 for males and 0.575 for females of Ny. intermedia and 0.649 for both sexes of Ny. neivai. The size of the populations in the five months ranged from 8,332-725,085 for Ny. intermedia males, 2,193-104,490 for Ny. intermedia females, 1,687-350,122 for Ny. neivai males and 254-49,705 for Ny. neivai females.

Survival, population pize, and gonotrophic cycle duration of Nyssomyia neivai (Diptera: Psychodidae) at an endemic area of American Cutaneous Leishmaniasis in Southeastern Brazil

The survival, absolute population size, gonotrophic cycle duration, and temporal and spatial abundance of Nyssomyia neivai (Pinto) were studied in a rural area endemic for American cutaneous leishmaniasis (ACL) in Conchal, Sõo Paulo State, southeastern Brazil, using mark-release-recapture techniques and by monitoring population fluctuation. The monthly abundance exhibited a unimodal pattern, with forest and domicile habitats having the highest relative abundances. A total of 1,873 males and 3,557 females were marked and released during the six experiments, of which 4.1-13.0 per cent of males and 4.1-11.8 per cent of females were recaptured. Daily survivorship estimated from the decline in recaptures per day was 0.681 for males and 0.667 for females. Gonotrophic cycle duration was estimated to be 4.0 d. Absolute population size was calculated using the Lincoln Index and ranged from 861 to 4,612 males and from 2,187 to 19,739 females. The low proportion of females that reach the age when they are potentially infective suggests that N. neivai has a low biological capacity to serve as a vector and that factors such as high biting rates and opportunistic feeding behavior would be needed to enable Leishmania (Viannia) braziliensis Vianna transmission. This agreed with the epidemiological pattern of ACL in southeastern Brazil that is characterized by low incidence, with isolated cases acquired principally within domiciliary habitats

The relationship between birth weight and insulin resistance in childhood

Chronic diseases that are typical of adulthood may originate in intra-uterine life through inadequate fetal development. The present epidemiological cohort study of 506 healthy children aged 5\20138 years evaluated the relationship between birth weight and insulin resistance in an age group that has been assessed in few similar studies. Insulin concentration was determined by chemiluminescence and insulin resistance by the homeostasis model assessment (HOMA). Blood glucose, total cholesterol and fractions (LDL cholesterol and HDL cholesterol) and TAG concentrations were determined by automated enzymatic methods. Linear regression analysis investigated the relationship between birth weight (assessed as a continuous variable and in three categories: small for gestational age, SGA; adequate for gestational age and large for gestational age) and the HOMA index, using backward stepwise selection and biological models to explain the causal pathway of the relationship. There were negativeassociations between birth weight (P < 0·001), SGA (P = 0·027) and the HOMA index, and a positive association between waist circumference (P < 0·001) and the HOMA index. Considering the significant associations between birth weight and waist circumference (P < 0·001) and waist circumference and insulin resistance (P < 0·001), we can probably suspect that lower birth weight is a common cause of higher waist circumference and insulin resistance. In summary, the results of the present study showed increased insulin resistance in apparently healthy, young children, who had lower weight at birth and higher measurements of waist circumference. There is a need to develop public health policies that adopt preventive measures to promote adequate maternal-fetal and child development and enable early diagnosis of metabolic abnormalities

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes. Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries. Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues. Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

Background: Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results: The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion: In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs.

Sugarcane genes associated with sucrose content

Background -: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results -: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion -: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

ProbFAST: Probabilistic Functional Analysis System Tool

Background: The post-genomic era has brought new challenges regarding the understanding of the organization and function of the human genome. Many of these challenges are centered on the meaning of differential gene regulation under distinct biological conditions and can be performed by analyzing the Multiple Differential Expression (MDE) of genes associated with normal and abnormal biological processes. Currently MDE analyses are limited to usual methods of differential expression initially designed for paired analysis. Results: We proposed a web platform named ProbFAST for MDE analysis which uses Bayesian inference to identify key genes that are intuitively prioritized by means of probabilities. A simulated study revealed that our method gives a better performance when compared to other approaches and when applied to public expression data, we demonstrated its flexibility to obtain relevant genes biologically associated with normal and abnormal biological processes. Conclusions: ProbFAST is a free accessible web-based application that enables MDE analysis on a global scale. It offers an efficient methodological approach for MDE analysis of a set of genes that are turned on and off related to functional information during the evolution of a tumor or tissue differentiation. ProbFAST server can be accessed at http://gdm.fmrp.usp.br/probfast.

Chromatographic analysis of lipid fractions in healthy dogs and dogs with obesity or hyperadrenocorticism

Obesity and endogenous hyperadrenocorticism (HAC) are common clinical conditions in veterinary practice, and both conditions have clinical and laboratory similarities, Such as weight gain and dyslipidemia. The objective of the present study was to characterize and compare the lipid profiles and plasma lipoprotein fractions in healthy dogs (n = 10), in obese dogs (n = 10), and in dogs with HAC (n = 6). All of the dogs were client owned. The lipoproteins were separated by fast protein liquid chromatography, and the plasma concentrations of total cholesterol and total triacylglycerol (TAG) were determined by enzymatic methods. When compared with the healthy and obese groups, dogs with HAC had a significant increase (P < 0.01) in the total concentrations of TAGs and cholesterol (CHOL), with higher distribution in the very low-density lipoprotein (VLDL)-CHOL fractions. In addition, the distributions of the high-density lipoprotein (HDL)-CHOL and HDL-TAG fractions were significantly lower (P < 0.01) in dogs with HAC than in healthy dogs. Considering the animals in this study, it was determined that the dogs with HAC differed significantly from the healthy and obese dogs regarding the metabolism of CHOL and TAG, as well as their VLDL and HDL fractions. Similar laboratory findings could allow veterinarians to distinguish obese dogs from those with HAC. In addition, dogs with HAC may be at higher risk for developing metabolic and atherosclerotic complications.

An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus

Background: Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R. sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences. Results: Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained similar to 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as ""unknown function"". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library. Conclusions: Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Analysis of genomic and functional RFLP derived markers associated with sucrose content, fiber and yield QTLs in a sugarcane (Saccharum spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.