BMC Research Notes - Latest Articles

Comparison of threshold selection methods for microarray gene co-expression matrices

Bhavesh Borate — 2009-12-02T00:00:00Z

Background: Network and clustering analyses of microarray co-expression correlation data often require application of a threshold to discard small correlations, thus reducing computational demands and decreasing the number of uninformative correlations. This study investigated threshold selection in the context of combinatorial network analysis of transcriptome data.FindingsSix conceptually diverse methods - based on number of maximal cliques, correlation of control spots with expressed genes, top 1% of correlations, spectral graph clustering, Bonferroni correction of p-values, and statistical power - were used to estimate a correlation threshold for three time-series microarray datasets. The validity of thresholds was tested by comparison to thresholds derived from Gene Ontology information. Stability and reliability of the best methods were evaluated with block bootstrapping.Two threshold methods, number of maximal cliques and spectral graph, used information in the correlation matrix structure and performed well in terms of stability. Comparison to Gene Ontology found thresholds from number of maximal cliques extracted from a co-expression matrix were the most biologically valid. Approaches to improve both methods were suggested. Conclusions: Threshold selection approaches based on network structure of gene relationships gave thresholds with greater relevance to curated biological relationships than approaches based on statistical pair-wise relationships.

Pyrosequencing identification of Mycobacterium tuberculosis W-Beijing

Zoheira Djelouadji — 2009-12-02T00:00:00Z

Background: The worldwide expanding Mycobacterium tuberculosis W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification.FindingsPyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 M. tuberculosis isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P inferior 10 to 5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (p=0.2, X chi -2 test). Pyrosequencing correctly identified M. tuberculosis organisms in 26/26 sputum specimens exhibiting acid-fact bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 Euro/test. Conclusions: Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.

Fas and FasL gene polymorphisms are not associated with cervical cancer but differ among Black and Mixed-ancestry South Africans

Koushik Chatterjee — 2009-11-26T00:00:00Z

Background: Cervical cancer is one of the most important cancers in African women. Polymorphisms in the Fas (FasR) and Fas ligand (FasL) genes have been reported to be associated with cervical cancer in certain populations. This study investigated whether these polymorphisms are associated with cervical cancer or human papillomavirus (HPV) infection in South African women.FindingsParticipants were 447 women with invasive cervical cancer (106 black African and 341 women of mixed-ancestry) and 424 healthy women controls, matched by age, (101 black African and 323 women of mixed-ancestry) and domicile (rural or urban). Two polymorphisms in Fas gene (FasR-1377G/A, FasR-670A/G) and one in FasL gene (FasL844T/C) were genotyped by TaqMan. None of the polymorphisms, or the Fas haplotypes, showed a significant association with cervical cancer. There was also no association with HPV infection in the control group. However, on analysis of the control group, highly significant allele, genotype and haplotype differences were found between the two ethnic groups. There were generally low frequencies of FasR-1377A alleles, FasR-670A alleles and FasL-844C alleles in black women compared to the women of mixed-ancestry. Conclusion: This is the first study on the role of Fas and FasL polymorphisms in cervical cancer in African populations. Our results suggest that these SNPs are not associated with cervical cancer in these populations. The allele frequencies of the three SNPs differed markedly between the indigenous African black and mixed-ancestry populations.

Thyroid hormones according to gestational age in pregnant Spanish women

Julia Pilar Bocos Terraz — 2009-11-26T00:00:00Z

Background: Thyroid function changes during pregnancy and maternal thyroid dysfunction have been associated with adverse outcomes. Our aim was to evaluate thyroid hormones levels in pregnant women resident in Aragon, Spain.FindingsSamples for 1198 pregnant women with no apparent thyroid disorders were analyzed, using paramagnetic microparticle and chemiluminescent detection technologies, in order to determine levels of thyroid stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), thyroid peroxidase antibodies (TPO-Ab), and thyroglobulin antibodies (Tg-Ab). Of the women in our sample, 85.22% had normal values for TPO-Ab and Tg-Ab and 14.77% had results revealing the presence of autoimmune diseases of the thyroid. The thyroid hormone reference values obtained according to gestational age (in brackets) were as follows: for free T3, values were 3.38 ± 0.52 pg/mL (<11 weeks), 3.45 ± 0.54 pg/mL (11-20 weeks), 3.32 ± 0.43 pg/mL (21-30 weeks), 3.21 ± 0.53 pg/mL (31-36 weeks), and 3.23 ± 0.41 pg/mL (>36 weeks); for free T4, values were 1.10 ± 0.14 ng/dL (<10 weeks), 1.04 ± 0.14 ng/dL (11-20 weeks), 0.93 ± 0.12 ng/dL (21-30 weeks), 0.90 ± 0.13 ng/dL (31-36 weeks), and 0.80 ± 0.21 ng/dL (>36 weeks); and for TSH, values were (μIU/mL): 1.12 ± 0.69 (<10 weeks), 1.05 ± 0.67 (11-20 weeks), 1.19 ± 0.60 (21-30 weeks), 1.38 ± 0.76 (31-36 weeks), and 1.46 ± 0.72 (>36 weeks). Conclusion: Pregnant women with normal antibody values according to gestational age had values for FT4 and TSH, but not for FT3, that differed to a statistically significant degree. The values we describe can be used as reference values for the Aragon region of Spain.

Effects of Indocyanine green on cultured retinal ganglion cells in-vitro

S Balaiya — 2009-11-25T00:00:00Z

Background: Indocyanine green (ICG) dye is commonly used to stain the inner limiting membrane during macular surgery. There are reports documenting the toxicity of ICG on retinal pigment epithelial cells, with conflicting results on retinal ganglion cells. In the present study, we evaluated the effect of ICG on retinal ganglion cells in vitro.Cultured rat retinal ganglion cells (RGC-5) were exposed to different concentrations of ICG (0.25, 0.5, 1.0, 1.25, & 5 mg/ml) for various time intervals (1, 5, 15, 30, & 60 minutes). Changes in structural morphology were identified using phase contrast bright field microscopy. Cell viability was quantified using the neutral red assay and cell death was characterized using Annexin-V staining.FindingsSignificant morphologic changes were observed at 15 and 60 minute intervals for all concentrations, where a reduction in cell size and loss of normal spindle shape was noted. A dose dependent decrease in cell viability was observed with increasing concentration of ICG as well as increasing exposure intervals. Compared to control, 48-74% reduction in neutral red uptake at all concentrations for exposures 5 minutes or greater (p<0.001). Even at 1 minute exposure, a dose dependent decline was observed in cell viability, with a 28-48% decline for doses above 1.25 mg/ml (p=0.007). Staining with Annexin-V, demonstrated a similar dose and time dependent increase in number of cells exhibiting early apoptosis. A greater than two-fold increase in Annexin-V expression for all doses at exposures greater than 1 minute was noted. Conclusion: ICG dye exhibits toxicity to retinal ganglion cells at clinically relevant doses after low exposure periods.

RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts

Joelle Vermeulen — 2009-11-25T00:00:00Z

Background: The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR.FindingsThe linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. Conclusion: Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.

Erythropoetin receptor expression in the human diabetic retina

Shaival Shah — 2009-11-25T00:00:00Z

Background: Recent evidence suggests erythropoietin (EPO) and the erythropoietin receptor (EPOR) may play a direct role in the pathogenesis of diabetic retinopathy. Better characterization of the EPO-EPOR signaling system in the ischemic retina may offer a new therapeutic modality for ischemic ophthalmic diseases. This study was performed to identify EPOR mRNA expression in the human diabetic eye.FindingsEPOR antisense RNA probes were validated on human pancreas tissue. In the normal eye, EPOR was expressed in the retinal ganglion cell layer. Minimal expression was observed in the inner and outer nuclear layer. Under conditions of diabetic retinopathy, EPOR expression shifted to photoreceptor cells. Increased expression was also observed in the peripheral retina. Conclusion: EPOR expression may be a biomarker or contribute to disease mechanisms in diabetic retinopathy.

Significant reduction in bacterial shedding and improvement in milk production in dairy farms after the use of a new inactivated paratuberculosis vaccine in a field trial

Ramon Juste — 2009-11-22T00:00:00Z

Background: Paratuberculosis vaccination has been in use in some regions for many decades, but results have not been widely spread. A new Mycobacterium avium subsp. paratuberculosis (MAP) killed vaccine was studied in relationship with its effects on fecal shedding and milk production in four farms while other two were kept as controls submitted to a test and cull scheme.FindingsFecal detection (n = 1829) and milking records (n = 2413) have been analyzed after two (5 herds) and four (1 herd) years of the beginning of the intervention. Shedder prevalence was reduced by 100% in three of the four vaccinated farms, 68% in the total of vaccinated animals and 46% in the two control farms. Total amount of MAP shed was reduced 77% in the vaccinated farms and 94% in the control farms. Overall milk production increased up to 3.9% after vaccination, while there was no significant difference in production after intervention in the non-vaccinated farms. Conclusion: MAP shedding reduction can be quickly accomplished both by vaccination and by testing and culling. However, vaccination appears to be a less expensive and more sustainable strategy since it required one single intervention and was also associated with an increase in milk production.

Traumatic Tympanic Membrane perforation: An aetiological profile

Olushola Afolabi — 2009-11-21T00:00:00Z

Background: Traumatic perforation of the tympanic membrane may be due to direct or indirect source. The aim of the study is to profile the various aetiologies of traumatic tympanic membrane perforation in Ilorin, north central Nigeria.A retrospective review of 64 patients seen at the University of Ilorin Teaching hospital, Ilorin, Nigeria over a ten year period (January 1998 to Dec 2007) with history of traumatic tympanic membrane perforation from various causes, these also included multiply injured patients with bleeding from middle ear as part of their presentations. The data retrieved included the biodata, the clinical presentations, source of injury, the clinical findings and the treatment outcome. The data were entered into an SPSS version 11 computer soft ware and analyzed descriptively.FindingsSixty four (64) ears were analysed, Age range 6 months to 50 yrs, mean age of 29.2 yrs 7.9% of them were ≤5 years, 29.7% between 21-34 years, and 37.7% were 35 years and above. The male to female ratio was 2.5:1.0. Commonest aetiology was from slaps, then road traffic injury (RTI) in 35.9% and 23.5%, Majority of the slap injury were from fights (30.5%), security agents, senior students and cultists at schools (17.4% each). Sudden hearing loss was a typical presentation (95.3%), majority of the patient defaulted from follow up once the symptoms of bleeding and pain subsided. Only 7.8% had neomembrane formation on follow up Conclusion: Traumatic perforation of the tympanic membrane is an uncommon injury that is under-reported, there is the need to educate on alternative punitive measure among students and security agents, unskilled removal of foreign body, early identification, evaluation and referral of patients reduces the attendant morbidity.

Zebrafish aplnra functions in epiboly

Svanhild Nornes — 2009-11-19T00:00:00Z

Background: The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown.FindingsBlockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord. Conclusion: aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.