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Abstract Background The Brassica species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to Arabidopsis thaliana, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of A. thaliana. A large number of EST sequences, derived from a range of different Brassica species, are available in the public database, but no public microarray resource has so far been developed for these species. Results We assembled unigenes using ~800,000 EST sequences, mainly from three species: B. napus, B. rapa and B. oleracea. The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in B. napus from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (i.e. the genes related by the ancestral genome triplication observed in diploid Brassica species). 90,864 unique sequence assemblies were developed. These were incorporated into the BAC sequence annotation for the Brassica rapa Genome Sequencing Project, enabling the identification of cognate genomic sequences for a proportion of them. A 60-mer oligo microarray comprising 94,558 probes was developed using the unigene sequences. Gene expression was analysed in reciprocal resynthesised B. napus lines and the B. oleracea and B. rapa lines used to produce them. The analysis showed that significant expression could consistently be detected in leaf tissue for 35,386 unigenes. Expression was detected across all four genotypes for 27,355 unigenes, genome-specific expression patterns were observed for 7,851 unigenes and 180 unigenes displayed other classes of expression pattern. Principal component analysis (PCA) clearly resolved the individual microarray datasets for B. rapa, B. oleracea and resynthesised B. napus. Quantitative differences in expression were observed between the resynthesised B. napus lines for 98 unigenes, most of which could be classified into non-additive expression patterns, including 17 that showed cytoplasm-specific patterns. We further characterized the unigenes for which A genome-specific expression was observed and cognate genomic sequences could be identified. Ten of these unigenes were found to be Brassica-specific sequences, including two that originate from complex loci comprising gene clusters. Conclusion We succeeded in developing a Brassica community microarray resource. Although expression can be measured for the majority of unigenes across species, there were numerous probes that reported in a genome-specific manner. We anticipate that some proportion of these will represent species-specific transcripts and the remainder will be the consequence of variation of sequences within the regions represented by the array probes. Our studies demonstrated that the datasets obtained from the arrays can be used for typical analyses, including PCA and the analysis of differential expression. We have also demonstrated that Brassica-specific transcripts identified in silico in the sequence assembly of public EST database accessions are indeed reported by the array. These would not be detectable using arrays designed using A. thaliana sequences.

We have demonstrated in canines that somatic nerve transfer to vesical branches of the inferior hypogastric plexus (IHP) can be used for bladder reinnervation after spinal root injury. Yet, the complex anatomy of the IHP hinders the clinical application of this repair strategy. Here, using human cadavers, we clarify the spatial relationships of the vesical branches of the IHP and nearby pelvic ganglia, with the ureteral orifice of the bladder. Forty-four pelvic regions were examined in 30 human cadavers. Gross post-mortem and intra-operative approaches (open anterior abdominal, manual laparoscopic, and robot-assisted) were used. Nerve branch distances and diameters were measured after thorough visual inspection and gentle dissection, so as to not distort tissue. The IHP had between 1 to 4 vesical branches (2.33 ± 0.72, mean ± SD) with average diameters of 0.51 ± 0.06 mm. Vesical branches from the IHP arose from a grossly visible pelvic ganglion in 93% of cases (confirmed histologically). The pelvic ganglion was typically located 7.11 ± 6.11 mm posterolateral to the ureteral orifice in 69% of specimens. With this in-depth characterization, vesical branches from the IHP can be safely located both posterolateral to the ureteral orifice and emanating from a more proximal ganglionic enlargement during surgical procedures.

Abstract The ToxCast in vitro screening program has provided concentration-response bioactivity data across more than a thousand assay endpoints for thousands of chemicals found in our environment and commerce. However, most ToxCast screening assays have evaluated individual biological targets in cancer cell lines lacking integrated physiological functionality (such as receptor signaling, metabolism). We evaluated differentiated HepaRGTM cells, a human liver-derived cell model understood to effectively model physiologically relevant hepatic signaling. Expression of 93 gene transcripts was measured by quantitative polymerase chain reaction using Fluidigm 96.96 dynamic arrays in response to 1060 chemicals tested in eight-point concentration-response. A Bayesian framework quantitatively modeled chemical-induced changes in gene expression via six transcription factors including: aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, androgen receptor, and peroxisome proliferator-activated receptor alpha. For these chemicals the network model translates transcriptomic data into Bayesian inferences about molecular targets known to activate toxicological adverse outcome pathways. These data also provide new insights into the molecular signaling network of HepaRGTM cell cultures.

Down syndrome (DS) is the most common genetic cause of intellectual disability and is primarily caused by the triplication of chromosome 21. The overexpression of amyloid precursor protein gene may be sufficient to drive Alzheimer's disease (AD) neuropathology that is observed in virtually all individuals with DS by the age of 40 years. There is relatively little information about inflammation in the DS brain and how the genetics of DS may alter inflammatory responses and modify the course of AD pathogenesis in this disorder. Using the macrophage classification system of M1, M2a, M2b, and M2c inflammatory phenotypes, we have shown that the early stages of AD are associated with a bias toward an M1 or M2a phenotype. In later stages of AD, markers of M1, M2a and M2c are elevated. We now report the inflammatory phenotype in a DS autopsy series to compare this with the progression in sporadic AD. Tissue from young DS cases (under 40 years of age, pre-AD) show a bias toward M1 and M2b states with little M2a or M2c observed. Older DS cases (over 40 with AD pathology) show a distinct bias toward an M2b phenotype. Importantly, this is distinct from sporadic AD where the M2b phenotype has been rarely, if ever observed in postmortem studies. Stimulated by immune complex activation of microglial cells and toll-like receptor activation, the M2b phenotype represents a unique neuroinflammatory state in diseased brain and may have significant implications for therapeutic intervention for persons with DS.

This study aimed to examine the trend of ambient air pollution (i.e., ozone (O3), nitrogen monoxide (NO), nitrogen dioxide (NO2), nitrogen oxides (NOx), sulfur dioxide (SO2), carbon monoxide (CO), benzene (C6H6) and particulate matter with an aerodynamic diameter smaller than 10 microns (PM10), and non-methane hydrocarbons (NMHCs) at 10 monitoring stations located in the main residential and industrial areas in the State of Kuwait over 6 years (2012–2017). We found that the SO2 level in industrial areas (0.065 ppm) exceeded the allowable range of SO2 in residential areas (0.030 ppm). Air pollution variables were defined by the Environmental Public Authority of Kuwait (K-EPA). In this study, integrated statistical analysis was performed to compare an established air pollution database to Kuwait Ambient Air Quality Guidelines and to determine the association between pollutants and meteorological factors. All pollutants were positively correlated, with the exception of most pollutants and PM10 and O3. Meteorological factors, i.e., the ambient temperature, wind speed and humidity, were also significantly associated with the above pollutants. Spatial distribution mapping indicated that the PM10 level remained high during the southwest monsoon (the hot and dry season), while the CO level was high during the northeast monsoon (the wet season). The NO2 and O3 levels were high during the first intermonsoon season.

Cell cycle arrest and stereotypic transcriptional responses to DNA damage induced by ionizing radiation (IR) were quantified in telomerase-expressing human diploid fibroblasts. Analysis of cytotoxicity demonstrated that 1.5 Gy IR inactivated colony formation by 40-45% in three fibroblast lines; this dose was used in all subsequent analyses. Fibroblasts exhibited > 90% arrest of progression from G2 to M at 2 hr post-IR and a similarly severe arrest of progression from G1 to S at 6 and 12 hr post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 hr post-IR caused the S and M compartments to empty by > 70% at 24 hr. Global gene expression was analyzed in IR-treated cells. A microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression that were common to the three fibroblast lines, including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, such as CDKN1A, GADD45, BTG2, and PLK3, were significantly up-regulated at 2 hr post-IR. Many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 hr post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts apparently as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile 24 hr post-IR with similarity to that of G0 growth quiescence. The results reveal a highly stereotypic pattern of response to IR in human diploid fibroblasts that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. [ABSTRACT FROM AUTHOR]

The toxic effects that organic solvents have on whole cells are important drawbacks in the application of these solvents in the production of fine chemicals by whole-cell stereoselective biotransformations. Although early studies found that organic solvents mainly destroyed the integrity of cell membranes by accumulating in the lipid bilayer of plasma membranes, the cellular metabolic responses to the presence of an organic solvent remain unclear. With the rapid development of genomics, it is possible to study cellular metabolism under perturbed conditions at the genome level. In this paper, the global gene expression profiles of Saccharomyces cerevisiae BY4743 grown in media with a high concentration of the organic solvent dimethyl sulfoxide (DMSO) were determined by microarray analysis of ∼6,200 yeast open reading frames (ORFs). From cells grown in SD minimal medium containing 1.0% (v/v) DMSO, changes in transcript abundance greater than or equal to 2.5-fold were classified. Genomic analyses showed that 1,338 genes were significantly regulated by the presence of DMSO in yeast. Among them, only 400 genes were previously found to be responsive to general environmental stresses, such as temperature shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. The DMSO-responsive genes were involved in a variety of cellular functions, including carbohydrate, amino acid and lipid metabolism, cellular stress responses, and energy metabolism. Most of the genes in the lipid biosynthetic pathways were down-regulated by DMSO treatment, whereas genes involved in amino acid biosynthesis were mostly up-regulated. The results demonstrate that the application of microarray technology allows better interpretation of metabolic responses, and the information obtained will be useful for the construction of engineered yeast strains with better tolerance of organic solvents. [ABSTRACT FROM AUTHOR]

Background: Epi proColon® is a new blood-based colorectal cancer (CRC) screening test designed to determine the methylation status of a promoter region of the SEPT9 (septin 9) gene in cell-free DNA isolated from plasma. We describe the analytical and clinical performance of the test., Methods: Analytical performance at 4 testing laboratories included determination of limit of detection, precision, and reproducibility of the SEPT9 test. Clinical performance was evaluated in a prospective study by use of samples (n = 1544) from subjects enrolled in the PRESEPT clinical trial. Results were analyzed by comparison with colonoscopy, the reference standard., Results: The limit of detection for methylated SEPT9 DNA was 7.8 pg/mL (95% CI 6-11 pg/mL) corresponding to <2 genome copies of methylated SEPT9 per milliliter of plasma. In the prospective clinical trial, sensitivity for all stages of CRC was 68% (95% CI 53%-80%) and for stage I-III CRC, 64% (48%-77%). Adjusted specificity, on the basis of negative colonoscopy findings, was 80.0% (78%-82%)., Significance: The Epi proColon test is a simple, real-time PCR-based assay for the detection of methylated SEPT9 DNA in blood that may provide a noninvasive CRC screening alternative for people noncompliant with current CRC screening guidelines., (© 2014 American Association for Clinical Chemistry.)

Background: With the price of next generation sequencing steadily decreasing, bacterial genome assembly is now accessible to a wide range of researchers. It is therefore necessary to understand the best methods for generating a genome assembly, specifically, which combination of sequencing and bioinformatics strategies result in the most accurate assemblies. Here, we sequence three E. coli strains on the Illumina MiSeq, Life Technologies Ion Torrent PGM, and Pacific Biosciences RS. We then perform genome assemblies on all three datasets alone or in combination to determine the best methods for the assembly of bacterial genomes., Results: Three E. coli strains - BL21(DE3), Bal225, and DH5α - were sequenced to a depth of 100× on the MiSeq and Ion Torrent machines and to at least 125× on the PacBio RS. Four assembly methods were examined and compared. The previously published BL21(DE3) genome [GenBank:AM946981.2], allowed us to evaluate the accuracy of each of the BL21(DE3) assemblies. BL21(DE3) PacBio-only assemblies resulted in a 90% reduction in contigs versus short read only assemblies, while N50 numbers increased by over 7-fold. Strikingly, the number of SNPs in PacBio-only assemblies were less than half that seen with short read assemblies (~20 SNPs vs. ~50 SNPs) and indels also saw dramatic reductions (~2 indel >5 bp in PacBio-only assemblies vs. ~12 for short-read only assemblies). Assemblies that used a mixture of PacBio and short read data generally fell in between these two extremes. Use of PacBio sequencing reads also allowed us to call covalent base modifications for the three strains. Each of the strains used here had a known covalent base modification genotype, which was confirmed by PacBio sequencing., Conclusion: Using data generated solely from the Pacific Biosciences RS, we were able to generate the most complete and accurate de novo assemblies of E. coli strains. We found that the addition of other sequencing technology data offered no improvements over use of PacBio data alone. In addition, the sequencing data from the PacBio RS allowed for sensitive and specific calling of covalent base modifications.

Background: The Brassica species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to Arabidopsis thaliana, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of A. thaliana. A large number of EST sequences, derived from a range of different Brassica species, are available in the public database, but no public microarray resource has so far been developed for these species., Results: We assembled unigenes using approximately 800,000 EST sequences, mainly from three species: B. napus, B. rapa and B. oleracea. The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in B. napus from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (i.e. the genes related by the ancestral genome triplication observed in diploid Brassica species). 90,864 unique sequence assemblies were developed. These were incorporated into the BAC sequence annotation for the Brassica rapa Genome Sequencing Project, enabling the identification of cognate genomic sequences for a proportion of them. A 60-mer oligo microarray comprising 94,558 probes was developed using the unigene sequences. Gene expression was analysed in reciprocal resynthesised B. napus lines and the B. oleracea and B. rapa lines used to produce them. The analysis showed that significant expression could consistently be detected in leaf tissue for 35,386 unigenes. Expression was detected across all four genotypes for 27,355 unigenes, genome-specific expression patterns were observed for 7,851 unigenes and 180 unigenes displayed other classes of expression pattern. Principal component analysis (PCA) clearly resolved the individual microarray datasets for B. rapa, B. oleracea and resynthesised B. napus. Quantitative differences in expression were observed between the resynthesised B. napus lines for 98 unigenes, most of which could be classified into non-additive expression patterns, including 17 that showed cytoplasm-specific patterns. We further characterized the unigenes for which A genome-specific expression was observed and cognate genomic sequences could be identified. Ten of these unigenes were found to be Brassica-specific sequences, including two that originate from complex loci comprising gene clusters., Conclusion: We succeeded in developing a Brassica community microarray resource. Although expression can be measured for the majority of unigenes across species, there were numerous probes that reported in a genome-specific manner. We anticipate that some proportion of these will represent species-specific transcripts and the remainder will be the consequence of variation of sequences within the regions represented by the array probes. Our studies demonstrated that the datasets obtained from the arrays can be used for typical analyses, including PCA and the analysis of differential expression. We have also demonstrated that Brassica-specific transcripts identified in silico in the sequence assembly of public EST database accessions are indeed reported by the array. These would not be detectable using arrays designed using A. thaliana sequences.

This report details the standardized experimental design and the different data streams that were collected (histopathology, clinical chemistry, hematology and gene expression from the target tissue (liver) and a bio-available tissue (blood)) after treatment with eight known hepatotoxicants (at multiple time points and doses with multiple biological replicates). The results of the study demonstrate the classification of histopathological differences, likely reflecting differences in mechanisms of cell-specific toxicity, using either liver tissue or blood transcriptomic data.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.