BMC Research Notes - Most viewed articles

Meta-analysis of SUMO1

Brian J Wilson — 2008-07-31

An abundantly growing body of literature implicates conjugation of SUMO in the regulation of many proteins and processes, yet the regulation of SUMO pathways is poorly understood. To gain insight into the players in the SUMO1 pathway I have performed an in-silico co-expression meta-analysis of SUMO1, comparing many different multi-microarray studies of various normal and human tumour tissues, from the Oncomine database. This serves as a data-driven predictor of pathway partners of SUMO1. While the data obtained need to be confirmed by future independent experiments and can currently only be considered a hypothesis, results implicate defender against cell death (DAD1) and the anti-apoptotic DEK oncogene as new pathway partners of SUMO1.

GPCRTree: online hierarchical classification of GPCR function

2008-08-21

Background: G protein-coupled receptors (GPCRs) play important physiological roles transducing extracellular signals into intracellular responses. Approximately 50% of all marketed drugs target a GPCR. There remains considerable interest in effectively predicting the function of a GPCR from its primary sequence.FindingsUsing techniques drawn from data mining and proteochemometrics, an alignment-free approach to GPCR classification has been devised. It uses a simple representation of a protein's physical properties. GPCRTree, a publicly-available internet server, implements an algorithm that classifies GPCRs at the class, sub-family and sub-subfamily level. Conclusions: A selective top-down classifier was developed which assigns sequences within a GPCR hierarchy. Compared to other publicly available GPCR prediction servers, GPCRTree is considerably more accurate at every level of classification. The server has been available online since March 2008 at URL: http://igrid-ext.cryst.bbk.ac.uk/gpcrtree/.

Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells

2008-07-28

Background -Rabbits provide an excellent model for many animal and human diseases, such as cardiovascular diseases, for the development of new vaccines in wound healing management and in the field of tissue engineering of tendon, cartilage, bone and skin.The study presented herein aims to investigate the biological properties of bone marrow rabbit MSCs cultured in different conditions, in order to provide a basis for their clinical applications in veterinary medicine.Findings -MSCs were isolated from 5 New Zealand rabbits. Fold increase, CFU number, doubling time, differentiation ability and immunophenotype were analyzed.With the plating density of 10 cells/cm2 the fold increase was significantly lower with DMEM-20%FCS and MSCs growth was significantly higher with αMEM-hEGF. The highest clonogenic ability was found at 100 cell/cm2 with MSCBM and at 10 cell/cm2 with M199. Both at 10 and 100 cells/cm2, in αMEM medium, the highest CFU increase was obtained by adding bFGF. Supplementing culture media with 10%FCS-10%HS determined a significant increase of CFU.Conclusion -Our data suggest that different progenitor cells with differential sensitivity to media, sera and growth factors exist and the choice of culture conditions has to be carefully considered for MSC management.

Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry

2008-08-28

Background: Circulating endothelial cells (CEC) and endothelial precursor cells (CEP) have been suggested as surrogate markers for angiogenesis and response to antiangiogenic therapy in cancer. However, CEC/CEP represent a tiny and heterogeneous cell population, rendering their enumeration in peripheral blood samples difficult. CEC/CEP quantification by flow cytometry is currently the most commonly applied method but technically demanding and poorly standardized. Thus, we investigated whether quantitative PCR-based detection might overcome these limitations. Methods: To test the sensitivity of both assays autologous endothelial colony forming cell clones (ECFC) and cord blood derived CD45- CD34+ progenitor cells were spiked into peripheral blood mononuclear cells (PBMNC) of healthy volunteers. Expression of CD45, CD31, CD34, KDR and CD133 was analyzed by 4-color flow cytometry. Furthermore, absolute gene expression of progenitor and endothelial cell markers was quantified in spiked samples, PBMNC from healthy controls (n=30), newly-diagnosed locally advanced rectal cancer patients (n=20) and patients with metastatic non-small cell lung cancer (NSCLC, n=25). Results: Applying flow cytometry, spiked ECFC and progenitor cells were detectable only at frequencies 0.01%, whereas using qPCR a detection limit of 0.001% was achievable. Moreover, qPCR revealed significantly increased gene transcripts of CD34 (p=0.028) and KDR (p=0.002) in patients with metastatic NSCLC but not in patients with locally advanced rectal cancer. Conclusions: QPCR may overcome the limitations of flow cytometry for the detection of CEC/CEP. However, further studies using qPCR-based assays as well as more specific CEC/CEP markers are needed to validate and improve the detection of these rare cell types.

A new approach to primer design for the control of PCR bias in methylation studies

Tomasz K Wojdacz, Lise Lotte Hansen and Alexander Dobrovic — 2008-07-28

Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols.

Streptococcus pneumoniae early response genes to human lung epithelial cells

Xin-Ming Song, Wayne Connor, Karsten Hokamp, Lorne A Babiuk and Andrew A Potter — 2008-08-12

Background: Streptococcus pneumoniae infection starts from colonization of the host respiratory tract where interaction with host respiratory tract epithelial cells occurs. To investigate pneumococcal genes that are involved in the early stage of interaction with host epithelial cells, transcriptional responses of an encapsulated pathogenic pneumococcal strain TIGR4 upon exposure to human lung epithelial cells A549 for 0.5 h and 1 h time periods were investigated by using TIGR (JCVI) microarray technology. Gene expression changes were validated by quantitative real-time PCR (qRT-PCR) analysis.FindingsWe observed different transcriptional profiles at two incubation time periods in which most gene expressions were down-regulated at 0.5 h but up-regulated at 1 h. Many genes associated with ribonucleotide biosynthesis were down-regulated at both time points, whereas the genes associated with cell envelope, energy metabolism, transport and protein synthesis were mostly up-regulated at 1 h. Furthermore, these profiles were compared to the transcriptomes of a TIGR4-derived strain in response to human macrophages for the same time periods. We found one set of genes that exhibited similar expression changes upon exposure to both types of host cells, including cell envelope-associated bgaA (SP0648) and nanA (SP1693), and uncharacterized gene clusters such as SP1677–SP1680 and SP1688–SP1690. Conclusion: These data indicate that at the early stage of interaction with host epithelial cells, a complex gene regulation and expression change occur in bacteria. Some of them might play an essential role during pathogen-host interactions and for the establishment of infection.

Educational inequalities in self-reported health in a general Iranian population

Ali Montazeri, Azita Goshtasebi and Mariam Vahdaninia — 2008-07-21

Background: The aim of this study was to investigate the relationship between educational level and self-reported health in an Iranian population, in order to provide evidence on social inequalities in health from a country in which such data need to be collected. Methods: This population-based study was carried out in Tehran, Iran. Individuals aged 15 years and over were interviewed. Self-reported health was measured by asking each individual to respond to the question: "In general how would you describe your health at present?" We used years of formal education as a measure of socioeconomic status and categorized the answers in five levels. Logistic regression analysis was used to estimate odds ratios and 95% confidence intervals indicating the contribution of educational level to self-reported health, adjusting for age, gender, marital status, and chronic diseases. Results: In all, 4163 individuals were interviewed. The mean age of the respondents was 35.1 years (SD = 16.0); 52% were female; the mean duration of formal education was 10.0 years (SD = 4.5); and 31% rated their health 'less than good'. Overall, women rated their health more poorly than men (P < 0.0001), and the findings showed that those with higher education rated their health significantly better than those with lower educational levels after adjusting for the age, gender, marital status and chronic diseases. The odds ratio for having 'less than good' self-rated health in those at the lowest educational level compared with those at the highest was 2.65 (95% CI = 1.88–3.73). Conclusion: The findings indicated an inverse relationship between educational level and self-rated health, and that age, gender, and chronic conditions had independent effects on self-reported health status. The findings of this first study from Iran suggest that health inequalities in developing countries such as Iran need to be addressed and policies for tackling the problem should be considered. In this respect, less well-educated people and women should be seen as the first target populations. It seems that although expanding the educational system might help the state to provide people with more educational options, it is also necessary to ensure that equal opportunities and access to quality education are provided for those from lower socioeconomic backgrounds; otherwise the current situation might cost the government more in the long term because of poor health among disadvantaged groups.

Whole genome amplification and its impact on CGH array profiles

2008-07-29

Background: Some array comparative genomic hybridisation (array CGH) platforms require a minimum of micrograms of DNA for the generation of reliable and reproducible data. For studies where there are limited amounts of genetic material, whole genome amplification (WGA) is an attractive method for generating sufficient quantities of genomic material from miniscule amounts of starting material. A range of WGA methods are available and the multiple displacement amplification (MDA) approach has been shown to be highly accurate, although amplification bias has been reported. In the current study, WGA was used to amplify DNA extracted from whole blood. In total, six array CGH experiments were performed to investigate whether the use of whole genome amplified DNA (wgaDNA) produces reliable and reproducible results. Four experiments were conducted on amplified DNA compared to unamplified DNA and two experiments on unamplified DNA compared to unamplified DNA.FindingsAll the experiments involving wgaDNA resulted in a high proportion of losses and gains of genomic material. Previously, amplification bias has been overcome by using amplified DNA in both the test and reference DNA. Our data suggests that this approach may not be effective, as the gains and losses introduced by WGA appears to be random and are not reproducible between different experiments using the same DNA. Conclusion: In light of these findings, the use of both amplified test and reference DNA on CGH arrays may not provide an accurate representation of copy number variation in the DNA.

Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants

Haitian Zhao, Ute Dreses-Werringloer, Peter Davies and Philippe Marambaud — 2008-06-30

Background: Cell cultures have become an indispensable tool in Alzheimer's disease research for studying amyloid-β (Aβ) metabolism. It is estimated that up to 35% of cell cultures in current use are infected with various mycoplasma species. In contrast with common bacterial and fungal infections, contaminations of cell cultures with mycoplasmas represent a challenging issue in terms of detectability and prevention. Mycoplasmas are the smallest and simplest self-replicating bacteria and the consequences of an infection for the host cells are variable, ranging from no apparent effect to induction of apoptosis.FindingsHere we present evidence that mycoplasmas from a cell culture contamination are able to efficiently and rapidly degrade extracellular Aβ. As a result, we observed no accumulation of Aβ in the conditioned medium of mycoplasma-positive cells stably transfected with the amyloid-β precursor protein (APP). Importantly, eradication of the mycoplasma contaminant – identified as M. hyorhinis – by treatments with a quinolone-based antibiotic, restored extracellular Aβ accumulation in the APP-transfected cells. Conclusion: These data show that mycoplasmas degrade Aβ and thus may represent a significant source of variability when comparing extracellular Aβ levels in different cell lines. On the basis of these results, we recommend assessment of mycoplasma contaminations prior to extracellular Aβ level measurements in cultured cells.

The complete chloroplast genome sequence of Brachypodium distachyon: sequence comparison and phylogenetic analysis of eight grass plastomes

2008-07-31

Background: Wheat, barley, and rye, of tribe Triticeae in the Poaceae, are among the most important crops worldwide but they present many challenges to genomics-aided crop improvement. Brachypodium distachyon, a close relative of those cereals has recently emerged as a model for grass functional genomics. Sequencing of the nuclear and organelle genomes of Brachypodium is one of the first steps towards making this species available as a tool for researchers interested in cereals biology.FindingsThe chloroplast genome of Brachypodium distachyon was sequenced by a combinational approach using BAC end and shotgun sequences derived from a selected BAC containing the entire chloroplast genome. Comparative analysis indicated that the chloroplast genome is conserved in gene number and organization with respect to those of other cereals. However, several Brachypodium genes evolve at a faster rate than those in other grasses. Sequence analysis reveals that rice and wheat have a ~2.1 kb deletion in their plastid genomes and this deletion must have occurred independently in both species. Conclusion: We demonstrate that BAC libraries can be used to sequence plastid, and likely other organellar, genomes. As expected, the Brachypodium chloroplast genome is very similar to those of other sequenced grasses. The phylogenetic analyses and the pattern of insertions and deletions in the chloroplast genome confirmed that Brachypodium is a close relative of the tribe Triticeae. Nevertheless, we show that some large indels can arise multiple times and may confound phylogenetic reconstruction.