Quality control in microarray assessment of gene expression in human airway epithelium

<p>Abstract</p> <p>Background</p> <p>Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) ≥ 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤ 3.0; and (3) the multi-chip normalization scaling factor ≤ 10.0.</p> <p>Results</p> <p>Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 ± 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 ± 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria.</p> <p>Conclusion</p> <p>Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.</p

Validation of thermal-mechanical modeling of stainless steel forgings

A constitutive model for recrystallization has been developed within the framework of an existing dislocation-based rate and temperature-dependent plasticity model. The theory has been implemented and tested in a finite element code. Material parameters were fit to data from monotonic compression tests on 304L steel for a wide range of temperatures and strain rates. The model is then validated by using the same parameter set in predictive thermal-mechanical simulations of experiments in which wedge forgings were produced at elevated temperatures. Model predictions of the final yield strengths compare well to the experimental results

Evolution of Hominin Polyunsaturated Fatty Acid Metabolism: From Africa to the New World

The metabolic conversion of dietary omega-3 and omega-6 18 carbon (18C) to long chain (>20 carbon) polyunsaturated fatty acids (LC-PUFAs) is vital for human life. The rate-limiting steps of this process are catalyzed by fatty acid desaturase (FADS) 1 and 2. Therefore, understanding the evolutionary history of the FADS genes is essential to our understanding of hominin evolution. The FADS genes have two haplogroups, ancestral and derived, with the derived haplogroup being associated with more efficient LC-PUFA biosynthesis than the ancestral haplogroup. In addition, there is a complex global distribution of these haplogroups that is suggestive of Neanderthal introgression. We confirm that Native American ancestry is nearly fixed for the ancestral haplogroup, and replicate a positive selection signal in Native Americans. This positive selection potentially continued after the founding of the Americas, although simulations suggest that the timing is dependent on the allele frequency of the ancestral Beringian population. We also find that the Neanderthal FADS haplotype is more closely related to the derived haplogroup and the Denisovan clusters closer to the ancestral haplogroup. Furthermore, the derived haplogroup has a time to the most recent common ancestor of 688,474years before present. These results support an ancient polymorphism, as opposed to Neanderthal introgression, forming in the FADS region during the Pleistocene with possibly differential selection pressures on both haplogroups. The near fixation of the ancestral haplogroup in Native American ancestry calls for future studies to explore the potential health risk of associated low LC-PUFA levels in these populations.Center for Health Related Informatics and Biomaging at the University of Maryland School of Medicine; National Institutes of Health/National Heart, Lung, and Blood Institute [U01 HL72518, HL087698, HL112064]; National Institutes of Health [R01-AT008621]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Helicobacter pylori Infection Promotes Methylation and Silencing of Trefoil Factor 2, Leading to Gastric Tumor Development in Mice and Humans

Background & Aims Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. Methods We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130[superscript F/F] mouse model of gastric cancer. Results In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130[superscript F/F]/Tff2[superscript −/−] mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130[superscript F/F]controls. The fundus of gp130[superscript F/F]/Tff2[superscript −/−] mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. Conclusions TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.National Health and Medical Research Council (Australia

Disease prevention strategies for QX disease (Marteilia sydneyi) of Sydney rock oysters (Saccostrea glomerata)

The Sydney rock oyster (Saccostrea glomerata) forms the basis of an important aquaculture industry on the east coast of Australia. During the 1970s, production of S. glomerata began to decline, in part as a result of mortalities arising from Queensland unknown (QX) disease. Histological studies implicated the paramyxean parasite Marteilia sydneyi in the disease outbreaks. Disease zoning was implemented to prevent the spread of M. sydneyi-infected oysters. This control measure hindered rock oyster farming, which historically has relied on transferring wild-caught spat between estuaries for on-growing to market size and has not prevented the subsequent occurrence of QX disease in the Georges and Hawkesbury rivers in central New South Wales. Management of QX disease has been hampered by the complicated life cycle of M. sydneyi, with outbreaks of QX disease likely to be regulated by a combination of the abundance of intermediate host of M. sydneyi, environmental stressors, and the immunocompetence of S. glomerata. The future of the Sydney rock oyster industry relies on understanding these factors and progressing the industry from relying on farming wild-caught seed to the successful commercialization of hatchery-produced QX-resistant S. glomerata

DNA polymerase δ-dependent repair of DNA single strand breaks containing 3′-end proximal lesions

Base excision repair (BER) is the major pathway for the repair of simple, non-bulky lesions in DNA that is initiated by a damage-specific DNA glycosylase. Several human DNA glycosylases exist that efficiently excise numerous types of lesions, although the close proximity of a single strand break (SSB) to a DNA adduct can have a profound effect on both BER and SSB repair. We recently reported that DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB are resistant to DNA glycosylase activity and this study further examines the processing of these ‘complex’ lesions. We first demonstrated that the damaged base should be excised before SSB repair can occur, since it impaired processing of the SSB by the BER enzymes, DNA ligase IIIα and DNA polymerase β. Using human whole cell extracts, we next isolated the major activity against DNA lesions located as a second nucleotide 5′-upstream to a DNA SSB and identified it as DNA polymerase δ (Pol δ). Using recombinant protein we confirmed that the 3′-5′-exonuclease activity of Pol δ can efficiently remove these DNA lesions. Furthermore, we demonstrated that mouse embryonic fibroblasts, deficient in the exonuclease activity of Pol δ are partially deficient in the repair of these ‘complex’ lesions, demonstrating the importance of Pol δ during the repair of DNA lesions in close proximity to a DNA SSB, typical of those induced by ionizing radiation

Identifying key drivers of the impact of an HIV cure intervention in sub-Saharan Africa

BACKGROUND:  The properties required of an intervention that results in eradication or control of HIV in absence of antiretroviral therapy (ART-free viral suppression) to make it cost-effective in low income settings are unknown. METHODS:  We used a model of HIV and ART to investigate the effect of introducing an ART-free viral suppression intervention in 2022 in an example country of Zimbabwe. We assumed that the intervention (cost: $500) would be accessible for 90$300 million (8.7% saving). An intervention of this efficacy costing anything up to $1400 is likely to be cost-effective in this setting. CONCLUSION:  Interventions aimed at curing HIV have the potential to improve overall disease burden and to reduce costs. Given the effectiveness and cost of ART, such interventions would have to be inexpensive and highly effective

Multiplexed, High Density Electrophysiology with Nanofabricated Neural Probes

Extracellular electrode arrays can reveal the neuronal network correlates of behavior with single-cell, single-spike, and sub-millisecond resolution. However, implantable electrodes are inherently invasive, and efforts to scale up the number and density of recording sites must compromise on device size in order to connect the electrodes. Here, we report on silicon-based neural probes employing nanofabricated, high-density electrical leads. Furthermore, we address the challenge of reading out multichannel data with an application-specific integrated circuit (ASIC) performing signal amplification, band-pass filtering, and multiplexing functions. We demonstrate high spatial resolution extracellular measurements with a fully integrated, low noise 64-channel system weighing just 330 mg. The on-chip multiplexers make possible recordings with substantially fewer external wires than the number of input channels. By combining nanofabricated probes with ASICs we have implemented a system for performing large-scale, high-density electrophysiology in small, freely behaving animals that is both minimally invasive and highly scalable

Histone deacetylase adaptation in single ventricle heart disease and a young animal model of right ventricular hypertrophy.

BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies