Integration and visualization of systems biology data in context of the genome

Background: High-density tiling arrays and new sequencing technologies are generating rapidly increasing volumes of transcriptome and protein-DNA interaction data. Visualization and exploration of this data is critical to understanding the regulatory logic encoded in the genome by which the cell dynamically affects its physiology and interacts with its environment. Results: The Gaggle Genome Browser is a cross-platform desktop program for interactively visualizing high-throughput data in the context of the genome. Important features include dynamic panning and zooming, keyword search and open interoperability through the Gaggle framework. Users may bookmark locations on the genome with descriptive annotations and share these bookmarks with other users. The program handles large sets of user-generated data using an in-process database and leverages the facilities of SQL and the R environment for importing and manipulating data. A key aspect of the Gaggle Genome Browser is interoperability. By connecting to the Gaggle framework, the genome browser joins a suite of interconnected bioinformatics tools for analysis and visualization with connectivity to major public repositories of sequences, interactions and pathways. To this flexible environment for exploring and combining data, the Gaggle Genome Browser adds the ability to visualize diverse types of data in relation to its coordinates on the genome. Conclusions: Genomic coordinates function as a common key by which disparate biological data types can be related to one another. In the Gaggle Genome Browser, heterogeneous data are joined by their location on the genome to create information-rich visualizations yielding insight into genome organization, transcription and its regulation and, ultimately, a better understanding of the mechanisms that enable the cell to dynamically respond to its environment.

A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

Background: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/.S3T source code and datasets can also be downloaded from the aforementioned website.

Ultra-Deep Sequencing Reveals the microRNA Expression Pattern of the Human Stomach

Background: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. Methodology/Principal Findings: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. Conclusions/Significance: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

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.

Time course proteomic profiling of human myocardial infarction plasma samples: An approach to new biomarker discovery

Background: The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. Methods: Ten individuals with recent acute ischemic-type chest pain (< 12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T(0)) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T(2)), and the next four stages occurred at 12 h intervals after T(0). Individuals (n = 7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). Results: Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p < 0.05). The 2STEMI group, had similar to 85% fewer differently expressed protein peaks than those without previous history of AMI (76, p < 0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T(0)), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. Conclusions: Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery. (C) 2011 Elsevier B.V. All rights reserved.

Bioinformatics construction of the human cell surfaceome

Cell surface proteins are excellent targets for diagnostic and therapeutic interventions. By using bioinformatics tools, we generated a catalog of 3,702 transmembrane proteins located at the surface of human cells (human cell surfaceome). We explored the genetic diversity of the human cell surfaceome at different levels, including the distribution of polymorphisms, conservation among eukaryotic species, and patterns of gene expression. By integrating expression information from a variety of sources, we were able to identify surfaceome genes with a restricted expression in normal tissues and/or differential expression in tumors, important characteristics for putative tumor targets. A high-throughput and efficient quantitative real-time PCR approach was used to validate 593 surfaceome genes selected on the basis of their expression pattern in normal and tumor samples. A number of candidates were identified as potential diagnostic and therapeutic targets for colorectal tumors and glioblastoma. Several candidate genes were also identified as coding for cell surface cancer/testis antigens. The human cell surfaceome will serve as a reference for further studies aimed at characterizing tumor targets at the surface of human cells.

Meningiomas and schwannomas: Molecular subgroup classification found by expression arrays

Microarray gene expression profiling is a high-throughput system used to identify differentially expressed genes and regulation patterns, and to discover new tumor markers. As the molecular pathogenesis of meningiomas and schwannomas, characterized by NF2 gene alterations, remains unclear and suitable molecular targets need to be identified, we used low density cDNA microarrays to establish expression patterns of 96 cancer-related genes on 23 schwannomas, 42 meningiomas and 3 normal cerebral meninges. We also performed a mutational analysis of the NF2 gene (PCR, dHPLC, Sequencing and MLPA), a search for 22q LOH and an analysis of gene silencing by promoter hypermethylation (MS-MLPA). Results showed a high frequency of NF2 gene mutations (40%), increased 22q LOH as aggressiveness increased, frequent losses and gains by MLPA in benign meningiomas, and gene expression silencing by hypermethylation. Array analysis showed decreased expression of 7 genes in meningiomas. Unsupervised analyses identified 2 molecular subgroups for both meningiomas and schwannomas showing 38 and 20 differentially expressed genes, respectively, and 19 genes differentially expressed between the two tumor types. These findings provide a molecular subgroup classification for meningiomas and schwannomas with possible implications for clinical practice.

Interieukin-17 and Kidney Allograft Outcome

Acute rejection episodes (ARE) are important complications that involve the interplay between mechanisms that maintain graft tolerance and promote rejection. The proinflammatory cytokine interieukin-17 (IL-17) has been implicated in many conditions in humans and mice. In kidney transplant patients, the evaluation IL-17 levels has been performed in only a few patients. We performed a cross-sectional study correlating quantitative IL-17 levels and clinical outcomes. Patients and methods. We studied 19 specimens from biopsies performed in patients (n = 19) who received isolated kidney grafts. ARE signs were present in 9 (47%) patients who provide specimens; whereas, 10 (53%) others showed no signs of rejection. Eighteen healthy control sample IL-17 underwent measurement, all of which were performed by an enzyme-linked immunosorbent assay method. We assessed other factors, such as the recipients demographic data, cold ischemia time, HLA mismatches, time elapsed from transplantation to the biopsy, posttransplantation status, antibody panel, donor type, and immunosuppressive treatment. Results. IL-17 levels were clearly increased among samples derived from patients with ongoing rejection (125.7 +/- 27.06 pg/mL) in contrast, to the nonrejection group, (30 +/- 13.32 pg/mL) (P < .05). Healthy controls showed no detectable IL-17 levels. Conclusions. These findings suggested that IL-17 was important in the pathophysiology of acute kidney rejection.

The Genetics of Alzheimer`s Disease in Brazil: 10 Years of Analysis in a Unique Population

Alzheimer`s Disease (AD) is the most common type of dementia among the elderly, with devastating consequences for the patient, their relatives, and caregivers. More than 300 genetic polymorphisms have been involved with AD, demonstrating that this condition is polygenic and with a complex pattern of inheritance. This paper aims to report and compare the results of AD genetics studies in case-control and familial analysis performed in Brazil since our first publication, 10 years ago. They include the following genes/markers: Apolipoprotein E (APOE), 5-hidroxytryptamine transporter length polymorphic region (5-HTTLPR), brain-derived neurotrophin factor (BDNF), monoamine oxidase A (MAO-A), and two simple-sequence tandem repeat polymorphisms (DXS1047 and D10S1423). Previously unpublished data of the interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) genes are reported here briefly. Results from others Brazilian studies with AD patients are also reported at this short review. Four local families studied with various markers at the chromosome 21, 19, 14, and 1 are briefly reported for the first time. The importance of studying DNA samples from Brazil is highlighted because of the uniqueness of its population, which presents both intense ethnical miscegenation, mainly at the east coast, but also clusters with high inbreeding rates in rural areas at the countryside. We discuss the current stage of extending these studies using high-throughput methods of large-scale genotyping, such as single nucleotide polymorphism microarrays, associated with bioinformatics tools that allow the analysis of such extensive number of genetics variables, with different levels of penetrance. There is still a long way between the huge amount of data gathered so far and the actual application toward the full understanding of AD, but the final goal is to develop precise tools for diagnosis and prognosis, creating new strategies for better treatments based on genetic profile.

Under flow impedimetric measurements using magnetic particles for label-free detection affinity target

The necessity to adapt sensors based on electrochemical techniques for high throughput analysis control increases the interest to develop new analytical systems able to perform measurements under buffer now. In this report we explored the possibility of employing a new system to make impedimetric measurements to detect the interaction between proteins and small molecules. The well-known biotin-streptavidin interaction was adopted to evaluate the proposed assembly. This system allows us to perform experiments under flow. Magnetic beads functionalized with streptavidin were used and first characterized using AFM and FTIR. Non-faradic impedance spectroscopy allowed the detection of the biotin-streptavidin interaction. Using our new system and under a flow of PBS buffer, 5 10-5 M of biotin was detected with a stable signal. (c) 2007 Elsevier B.V. All rights reserved.

Fast Acquisition of (13)C NMR Spectra using the Steady-state Free Precession Sequence

In this paper, we show that the steady-state free precession sequence can be used to acquire (13)C high-resolution nuclear magnetic resonance spectra and applied to qualitative analysis. The analysis of brucine sample using this sequence with 60 degrees flip angle and time interval between pulses equal to 300 ms (acquisition time, 299.7 ms; recycle delay, 300 ms) resulted in spectrum with twofold enhancement in signal-to-noise ratio, when compared to standard (13)C sequence. This gain was better when a much shorter time interval between pulses (100 ms) was applied. The result obtained was more than fivefold enhancement in signal-to-noise ratio, equivalent to more than 20-fold reduction in total data recording time. However, this short time interval between pulses produces a spectrum with severe phase and truncation anomalies. We demonstrated that these anomalies can be minimized by applying an appropriate apodization function and plotting the spectrum in the magnitude mode.

Ultra-Fast Gradient LC Method for Omeprazole Analysis Using a Monolithic Column: Assay Development, Validation, and Application to the Quality Control of Omeprazole Enteric-Coated Pellets

A method was optimized for the analysis of omeprazole (OMZ) by ultra-high speed LC with diode array detection using a monolithic Chromolith Fast Gradient RP 18 endcapped column (50 x 2.0 mm id). The analyses were performed at 30 degrees C using a mobile phase consisting of 0.15% (v/v) trifluoroacetic acid (TFA) in water (solvent A) and 0.15% (v/v) TFA in acetonitrile (solvent B) under a linear gradient of 5 to 90% B in 1 min at a flow rate of 1.0 mL/min and detection at 220 nm. Under these conditions, OMZ retention time was approximately 0.74 min. Validation parameters, such as selectivity, linearity, precision, accuracy, and robustness, showed results within the acceptable criteria. The method developed was successfully applied to OMZ enteric-coated pellets, showing that this assay can be used in the pharmaceutical industry for routine QC analysis. Moreover, the analytical conditions established allow for the simultaneous analysis of OMZ metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in the same run, showing that this method can be extended to other matrixes with adequate procedures for sample preparation.