The Reproducibility of Lists of Differentially Expressed Genes in Microarray Studies

Reproducibility is a fundamental requirement in scientific experiments and clinical contexts. Recent publications raise concerns about the reliability of microarray technology because of the apparent lack of agreement between lists of differentially expressed genes (DEGs). In this study we demonstrate that (1) such discordance may stem from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion, the lists become much more reproducible, especially when fewer genes are selected; and (3) the instability of short DEG lists based on P cutoffs is an expected mathematical consequence of the high variability of the t-values. We recommend the use of FC ranking plus a non-stringent P cutoff as a baseline practice in order to generate more reproducible DEG lists. The FC criterion enhances reproducibility while the P criterion balances sensitivity and specificity

Comparative pulmonary toxicity of two Ceria nanoparticles with the same primary size

Ceria nanoparticles (nano-ceria) have recently gained a wide range of applications, which might pose unwanted risks to both the environment and human health. The greatest potential for the environmental discharge of nano-ceria appears to be in their use as a diesel fuel additive. The present study was designed to explore the pulmonary toxicity of nano-ceria in mice after a single exposure via intratracheal instillation. Two types of nano-ceria with the same distribution of a primary size (3–5 nm), but different redox activity, were used: Ceria-p, synthesized by a precipitation route, and Ceria-h, synthesized by a hydrothermal route. Both Ceria-p and Ceria-h induced oxidative stress, inflammatory responses and cytotoxicity in mice, but their toxicological profiles were quite different. The mean size of Ceria-p agglomerates was much smaller compared to Ceria-h, thereby causing a more potent acute inflammation, due to their higher number concentration of agglomerates and higher deposition rate in the deep lung. Ceria-h had a higher reactivity to catalyzing the generation of reactive oxygen species (ROS), and caused two waves of lung injury: bronchoalveolar lavage (BAL) inflammation and cytotoxicity in the early stage and redox-activity-evoked lipid peroxidation and pro-inflammation in the latter stage. Therefore, the size distribution of ceria-containing agglomerates in the exhaust, as well as their surface chemistry are essential characteristics to assess the potential risks of using nano-ceria as a fuel additive

Early Intervention of Didang Decoction on MLCK Signaling Pathways in Vascular Endothelial Cells of Type 2 Diabetic Rats

In the study, type 2 diabetic rat model was established using streptozotocin (STZ) combined with a high-fat diet, and the rats were divided into control and diabetic groups. Diabetic groups were further divided into nonintervening, simvastatin, Didang Decoction (DDD) early-phase intervening, DDD mid-phase intervening, and DDD late-phase intervening groups. The expression level of MLCK was detected using Western Blot analysis, and the levels of cyclic adenosine monophosphate (cAMP), protein kinase C (PKC), and protein kinase A (PKA) were examined using Real Time PCR. Under the electron microscope, the cells in the early-DDD-intervention group and the simvastatin group were significantly more continuous and compact than those in the diabetic group. Compared with the control group, the expression of cAMP-1 and PKA was decreased in all diabetic groups, whereas the expression of MLCK and PKC was increased in early- and mid-phase DDD-intervening groups (P<0.05); compared with the late-phase DDD-intervening group, the expression of cAMP-1 and PKA was higher, but the level of MLCK and PKC was lower in early-phase DDD-intervening group (P<0.05). In conclusion, the early use of DDD improves the permeability of vascular endothelial cells by regulating the MLCK signaling pathway

Polymethoxyflavones extracted from Bauhinia championii alleviate LPS-induced acute lung injury by ameliorating endoplasmic reticulum stress

BackgroundAcute lung injury (ALI), a critical respiratory condition, often escalates into acute respiratory distress syndrome, which is associated with significant morbidity and mortality. Bauhinia championii, a botanical drug used in traditional Chinese medicine, is reputed for its antioxidative and anti-hypoxia effects. However, the active metabolites within B. championii and their mechanisms of action in alleviating ALI remain to be elucidated.MethodsA comprehensive literature review and database search within Chemistry Database were conducted to compile a complete profile of the metabolites identified in B. championii. Utilizing network analysis, we predicted potential targets of metabolites in B. championii (MBC) for ALI treatment. A protein-protein interaction (PPI) network was constructed using Cytoscape 3. 9. 1, complemented by GO annotations and KEGG pathway enrichment analyses via the DAVID online platform. The isolation and characterization of polymethoxyflavones (PMFs) from B. championii were performed using HPLC and confirmed by LC-MS. In vivo pharmacological assessments were executed to substantiate the network analysis predictions. Moreover, the Autodock software facilitated molecular docking studies to elucidate the role of endoplasmic reticulum (ER) stress modulation in ALI treatment by PMFs.Results17 known MBC were identified in which 7 active metabolites of flavonoids were used as predictive targets. 122 target genes associated with both MBC and ALI were tested for KEGG and GO enrichment analyses, which indicated these target genes involvement in antioxidant, anti-inflammatory, and anti-apoptotic pathways. The PMFs were extracted from B. championii and identified as 5, 6, 7, 3′, 4′-pentamethoxyflavone, 5, 6, 7, 3′, 4′, 5′-hexamethoxyflavone, 5, 7, 3′, 4′, 5′-pentamethoxyflavone, 5, 6, 7, 5′-tetramethoxy-3′, 4′-methylenedioxyflavone and 5, 7, 5′-trimethoxy-3′, 4′-methylenedioxyflavone. PMFs were effective in alleviating LPS-induced pulmonary inflammatory responses for releasing ALI. In addition, PMFs inhibited the secretion of GSH-Px and CAT, reduced the accumulation of HYP and MDA as well as the infiltration of inflammatory cells, not to mention alleviated LPS-induced apoptosis by inhibiting the Caspase 3-mediated apoptosis pathway. Furthermore, the PMFs can spontaneously bind to multiple ER stress targets to exert the effect of calming ER stress to alleviate ALI.ConclusionPMFs inhibited the expression of inflammatory cytokines and reduced oxidative stress injury to resist apoptosis in lung. Moreover, PMFs attenuated LPS-induced ER stress activation by regulating ER stress related targets, which in turn alleviated ALI

Critical Epitopes in the Nucleocapsid Protein of SFTS Virus Recognized by a Panel of SFTS Patients Derived Human Monoclonal Antibodies

BACKGROUND: SFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection

Cross-platform comparability of microarray technology: Intra-platform consistency and appropriate data analysis procedures are essential

BACKGROUND: The acceptance of microarray technology in regulatory decision-making is being challenged by the existence of various platforms and data analysis methods. A recent report (E. Marshall, Science, 306, 630–631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676–5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology. RESULTS: We reanalyzed Tan's dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tan's study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. and Marshall. CONCLUSION: Our results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures. Thus, we are progressively coordinating the MAQC project, a community-wide effort for microarray quality control

Microarray scanner calibration curves: characteristics and implications

BACKGROUND: Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear. RESULTS: By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias. CONCLUSION: It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy

Assessing reproducibility of inherited variants detected with short-read whole genome sequencing

Background: Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. Results: To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30x. Conclusions: Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.Peer reviewe

Assessing Reproducibility of Inherited Variants Detected With Short-Read Whole Genome Sequencing

Background: Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. Results: To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when \u3e 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. Conclusions: Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS