Profiling alternatively spliced mRNA isoforms for prostate cancer classification

BACKGROUND: Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. RESULTS: As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR) of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM) classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. CONCLUSION: These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays

Molecular Investigation of the Transmission Pattern of Brucella suis 3 From Inner Mongolia, China

Brucellosis is an endemic disease in China affecting both humans and livestock. The aim of the present study was to analyze two Brucella strains isolated from sheep spleens from Ulanqab in Inner Mongolia, China using classical and molecular typing techniques. The two strains were identified as Brucella suis biovar 3 and were closely related to isolates previously obtained from two different hosts (human and swine) in Guangxi Province. Our results suggest that B. suis can be directly or indirectly transferred from swine to sheep, which act as reservoirs for B. suis infection and later transmitted to humans. Multiple locus variable-number tandem repeat analysis (MLVA) is a useful tool for tracing the geographical origin of brucellosis infections and elucidating its transmission patterns

Odabir sojeva bakterija mliječne kiseline sa sposobnošću proizvodnje egzopolisaharida izoliranih iz tradicionalnog jogurta regije Inner-Mongolija

Lactic acid bacteria (LAB) isolated from Inner Mongolian traditional yoghurt were evaluated for the production of exopolysaccharides (EPS) by phenol-sulphuric acid method after ethanol precipitation and dialysis. Total polysaccharide was extracted from sucrose-containing MRS broth cultures of the selected LAB strains. Comparison of the EPS yields revealed that among tested LAB, strain 37 exhibited the highest production of 536.904 mg/L. The strain was identified as Leuconostoc citreum with carbohydrate assimilation profiling, 16S rRNA and pheS gene sequencing. The Ln. citreum 37 was found to be a novel EPS producing strain. It was found that there was no direct linear relation between the colony size and EPS yield, so the colony size could not to be used to screen EPS-producing strains.U ovom radu ispitivana je sposobnost proizvodnje egzoplisaharida (EPS) u bakterija mliječne kiseline (BMK) izoliranih iz tradicionalnog jogurta regije Inner-Mongolija. Pri tom je nakon taloženja i dijalize etanola korištena metoda fenol-sumporne kiseline. Ukupni polisaharidi izolirani su iz MRS hranjivog bujona u kojemu su uz dodatak saharoze uzgajani odabrani sojevi BMK. Usporedba prinosa egzopolisaharida pokazala je da je među analiziranim sojevima BMK, soj označen brojem 37 imao najvišu sposobnost proizvodnje EPS i to u koncentraciji od 536.904 mg/L. Na temelju analize asimilacije različitih ugljikohidrata te sekvenciranja regije 16S rRNA i gena pheS soj je identificiran kao Leuconostoc citreum. Time je otkriven novi soj (Ln. citreum 37) sa sposobnošću proizvodnje EPS. Također je utvrđeno kako veličina kolonija nije u linearnoj korelaciji s prinosom EPS te se stoga ne može koristiti za nadzor sojeva sa sposobnošću proizvodnje EPS

Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples

Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples

Toxicity of Melaleuca alternifolia essential oil to the mitochondrion and NAD+/NADH dehydrogenase in Tribolium confusum

Background In our previous study, Melaleuca alternifolia essential oil (EO) was considered to have an insecticidal effect by acting on the mitochondrial respiratory chain in insects. However, the mode of action is not fully understood. Methods In this study, we investigated the insecticidal efficacy of the M. alternifolia EO against another major stored-product pest, Tribolium confusum Jacquelin du Val. Rarefaction and vacuolization of the mitochondrial matrix were evident in oil-fumigated T. confusum adults. Results Alterations to the mitochondria confirmed the insecticidal effect of the M. alternifolia EO. Furthermore, comparative transcriptome analysis of T. confusum using RNA-seq indicated that most of the differentially expressed genes were involved in insecticide detoxification and mitochondrial function. The biochemical analysis showed that the intracellular NAD+/NADH ratio is involved in the differential effect of the M. alternifolia EO. Discussion These results led us to conclude that NAD+/NADH dehydrogenase may be the prime target site for the M. alternifolia EO in insects, leading to blocking of the mitochondrial respiratory chain

Genomic Characterization Provides New Insights for Detailed Phage- Resistant Mechanism for Brucella abortus

As the causative agent of cattle brucellosis, Brucella abortus commonly exhibits smooth phenotype (by virtue of colony morphology) that is characteristically sensitive to specific Brucella phages, playing until recently a major role in taxonomical classification of the Brucella species by the phage typing approach. We previously reported the discrepancy between traditional phenotypic typing and MLVA results of a smooth phage-resistant (SPR) strain Bab8416 isolated from a 45-year-old custodial worker with brucellosis in a cattle farm. Here, we performed whole genome sequencing and further obtained a complete genome sequence of strain Bab8416 by a combination of multiple NGS technologies and routine PCR sequencing. The detailed genetic differences between B. abortus SPR Bab8416 and large smooth phage-sensitive (SPS) strains were investigated in a comprehensively comparative genomic study. The large indels between B. abortus SPS strains and Bab8416 showed possible divergence between two evolutionary branches at a far phylogenetic node. Compared to B. abortus SPS strain 9-941 (Bab9-941), the specific re-arrangement event in Bab8416 displaying a closer linear relationship with B. melitensis 16M than other B. abortus strains resulted in the truncation of c-di-GMP synthesis, and 3 c-di-GMP-metabolizing genes, were present in Bab8416 and B. melitensis 16M, but absent in Bab9-941 and other B. abortus strains, indicating potential SPR-associated key determinants and novel molecular mechanisms. Moreover, despite almost completely intact smooth LPS related genes, only one mutated OmpA family protein of Bab8416, functionally related to flagellar and efflux pump, was newly identified. Several point mutations were identified to be Bab8416 specific while a majority of them were verified to be B. abortus ST2 characteristic. In conclusion, our study therefore identifies new SPR-associated factors that could play a role in refining and updating Brucella taxonomic schemes and provides resources for further detailed analysis of mechanism for Brucella phage resistance

Molecular Characterization of Tb, a New Approach for an Ancient Brucellaphage

Tb (Tbilisi), the reference Brucellaphage strain, was classified as a member of the Podoviridae family with icosahedral capsids (57 ± 2 nm diameter) and short tails (32 ± 3 nm long). Brucellaphage DNA was double stranded and unmethylated; its molecular size was 34.5 kilobase pairs. Some sequences were found through RAPD analysis, TA cloning technology, and structural proteins were observed by using SDS-PAGE. Thus, the results have laid the foundation for the wider use of Brucellaphage’s basic mechanisms and practical applications

Genome-wide expression assay comparison across frozen and fixed postmortem brain tissue samples

<p>Abstract</p> <p>Background</p> <p>Gene expression assays have been shown to yield high quality genome-wide data from partially degraded RNA samples. However, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor RNA quality and other technical limitations inherent in many assays. We compared cDNA-mediated annealing, selection, and ligation (DASL)- and <it>in vitro </it>transcription (IVT)-based genome-wide expression profiling assays on RNA samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.</p> <p>Results</p> <p>The DASL-based platform produced expression results of greater reliability than the IVT-based platform in artificially degraded reference brain RNA and RNA from frozen tissue-based samples. Although data associated with a small sample of formalin-fixed RNA samples were poor when obtained from both assays, the DASL-based platform exhibited greater reliability in a subset of probes and samples.</p> <p>Conclusions</p> <p>Our results suggest that the DASL-based gene expression-profiling platform may confer some advantages on mRNA assays of the brain over traditional IVT-based methods. We ultimately consider the implications of these results on investigations of neuropsychiatric disorders.</p