Technical Variability Is Greater than Biological Variability in a Microarray Experiment but Both Are Outweighed by Changes Induced by Stimulation

INTRODUCTION: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS). METHODS AND RESULTS: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined. CONCLUSIONS: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases

Combined changes in Wnt signalling response and contact inhibition induce altered proliferation in radiation treated intestinal crypts

Curative intervention is possible if colorectal cancer is identified early, underscoring the need to detect the earliest stages of malignant transformation. A candidate biomarker is the expanded proliferative zone observed in crypts before adenoma formation, also found in irradiated crypts. However, the underlying driving mechanism for this is not known. Wnt signaling is a key regulator of proliferation, and elevated Wnt signaling is implicated in cancer. Nonetheless, how cells differentiate Wnt signals of varying strengths is not understood. We use computational modeling to compare alternative hypotheses about how Wnt signaling and contact inhibition affect proliferation. Direct comparison of simulations with published experimental data revealed that the model that best reproduces proliferation patterns in normal crypts stipulates that proliferative fate and cell cycle duration are set by the Wnt stimulus experienced at birth. The model also showed that the broadened proliferation zone induced by tumorigenic radiation can be attributed to cells responding to lower Wnt concentrations and dividing at smaller volumes. Application of the model to data from irradiated crypts after an extended recovery period permitted deductions about the extent of the initial insult. Application of computational modeling to experimental data revealed how mechanisms that control cell dynamics are altered at the earliest stages of carcinogenesis

Discrimination and Hate Crimes in the Context of Neighborhood Poverty and Stressors Among HIV-Positive African-American Men Who Have Sex with Men

In a sample of HIV-positive African-American men who have sex with men (MSM), we examined neighborhood factors that may contextualize perceived discrimination from three intersecting stigmatized characteristics: race, HIV status, and sexual orientation. HIV-positive African-American MSM (N = 162, mean age = 44, SD = 8) provided information on neighborhood-related stressors and discrimination experiences related to being Black, HIV-positive, or perceived as gay. Residential ZIP codes and US Census data were used to determine neighborhood poverty rates. Regressions, controlling for socio-demographics, indicated that (1) higher neighborhood poverty was significantly related to more frequent experiences with hate crimes (Gay-related: b = 1.15, SE = .43, p < .008); and (2) higher neighborhood-related stressors were significantly related to more frequent discrimination (Black-related: b = .91, SE = .28, p = .001; gay-related: b = .71, SE = .29, p = .01; and HIV-related: b = .65, SE = .28, p = .02) and hate crimes (Gay-related: b = .48, SE = .13, p = .001; and Black-related: b = .28, SE = .14, p = .04). For HIV-positive African-American MSM, higher neighborhood poverty and related stressors are associated with experiencing more discrimination and hate crimes. Interventions for this group should promote individual- and neighborhood-level socioeconomic empowerment and stigma reduction