Eugene Demidenko

Mixed models : theory and applications

Preface. 1. Introduction: Why Mixed Models? 2. MLE for LME Model. 3. Statistical Properties of the LME Model. 4. Growth Curve Model and Generalizations. 5. Meta--analysis Model. 6. Nonlinear Marginal Model. 7. Generalized Linear Mixed Models. 8. Nonlinear Mixed Effects Model. 9. Diagnostics and Influence Analysis. 10. Tumor Regrowth Curves. 11. Statistical Analysis of Shape. 12. Statistical Image Analysis. 13. Appendix: Useful Facts and Formulas. References. Index.

MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors

MicroRNAs (miRNAs) regulate gene expression. It has been suggested that obtaining miRNA expression profiles can improve classification, diagnostic, and prognostic information in oncology. Here, we sought to comprehensively identify the miRNAs that are overexpressed in lung cancer by conducting miRNA microarray expression profiling on normal lung versus adjacent lung cancers from transgenic mice. We found that miR-136, miR-376a, and miR-31 were each prominently overexpressed in murine lung cancers. Real-time RT-PCR and in situ hybridization (ISH) assays confirmed these miRNA expression profiles in paired normal-malignant lung tissues from mice and humans. Engineered knockdown of miR-31, but not other highlighted miRNAs, substantially repressed lung cancer cell growth and tumorigenicity in a dose-dependent manner. Using a bioinformatics approach, we identified miR-31 target mRNAs and independently confirmed them as direct targets in human and mouse lung cancer cell lines. These targets included the tumor-suppressive genes large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A), and expression of each was augmented by miR-31 knockdown. Their engineered repression antagonized miR-31-mediated growth inhibition. Notably, miR-31 and these target mRNAs were inversely expressed in mouse and human lung cancers, underscoring their biologic relevance. The clinical relevance of miR-31 expression was further independently and comprehensively validated using an array containing normal and malignant human lung tissues. Together, these findings revealed that miR-31 acts as an oncogenic miRNA (oncomir) in lung cancer by targeting specific tumor suppressors for repression.

Arsenic Exposure Is Associated with Decreased DNA Repair in Vitro and in Individuals Exposed to Drinking Water Arsenic

The mechanism(s) by which arsenic exposure contributes to human cancer risk is unknown; however, several indirect cocarcinogenesis mechanisms have been proposed. Many studies support the role of As in altering one or more DNA repair processes. In the present study we used individual-level exposure data and biologic samples to investigate the effects of As exposure on nucleotide excision repair in two study populations, focusing on the excision repair cross-complement 1 (ERCC1) component. We measured drinking water, urinary, or toenail As levels and obtained cryopreserved lymphocytes of a subset of individuals enrolled in epidemiologic studies in New Hampshire (USA) and Sonora (Mexico). Additionally, in corroborative laboratory studies, we examined the effects of As on DNA repair in a cultured human cell model. Arsenic exposure was associated with decreased expression of ERCC1 in isolated lymphocytes at the mRNA and protein levels. In addition, lymphocytes from As-exposed individuals showed higher levels of DNA damage, as measured by a comet assay, both at baseline and after a 2-acetoxyacetylaminofluorene (2-AAAF) challenge. In support of the in vivo data, As exposure decreased ERCC1 mRNA expression and enhanced levels of DNA damage after a 2-AAAF challenge in cell culture. These data provide further evidence to support the ability of As to inhibit the DNA repair machinery, which is likely to enhance the genotoxicity and mutagenicity of other directly genotoxic compounds, as part of a cocarcinogenic mechanism of action.

Incidence of transitional cell carcinoma of the bladder and arsenic exposure in New Hampshire

AbstractObjective: Arsenic is a known bladder carcinogen and populations exposed to high arsenic levels in their water supply have reported elevated bladder cancer mortality and incidence rates. To examine the effects of lower levels of arsenic exposure on bladder cancer incidence, we conducted a case–control study in New Hampshire, USA where levels above 10 μ/l are commonly found in private wells. Methods: We studied 383 cases of transitional cell carcinoma of the bladder cancer, newly diagnosed between July 1, 1994 and June 30, 1998 and 641 general population controls. Individual exposure to arsenic was determined in toenail clippings using instrumental neutron activation analysis. Results: Among smokers, an elevated odds ratio (OR) for bladder cancer was observed for the uppermost category of arsenic (OR: 2.17, 95% CI: 0.92–5.11 for greater than 0.330 mcg/g compared to less than 0.06 μ/g). Among never smokers, there was no association between arsenic and bladder cancer risk. Conclusions: These, and other data, suggest that ingestion of low to moderate arsenic levels may affect bladder cancer incidence, and that cigarette smoking may act as a co-carcinogen.

Life spans and senescent phenotypes in two strains of Zebrafish (Danio rerio)

Zebrafish have become a widely used model organism in developmental biology research. In order to initiate an experimental foundation for aging studies, we have determined some basic gerontological parameters for populations of outbred zebrafish, and the golden sparse strain. Outbred zebrafish manifested a mean life span of about 42 months, with the longest living individual surviving for 66 months. The golden sparse populations had a mean life span of 36 months and a maximum longevity of 58 months. Skeletal length at death increased with age, suggestive of indeterminate growth. A common age-related phenotype was spinal curvature. Radiographic analysis excluded bony changes as the cause of the spinal curvature, suggesting muscle abnormalities as a primary mechanism. These data and a growing abundance of related biological resources suggest that the zebrafish may be a compelling model organism for studies on aging.

Tumor cell targeting by iron oxide nanoparticles is dominated by different factors in vitro versus in vivo.

Realizing the full potential of iron oxide nanoparticles (IONP) for cancer diagnosis and therapy requires selective tumor cell accumulation. Here, we report a systematic analysis of two key determinants for IONP homing to human breast cancers: (i) particle size and (ii) active vs passive targeting. In vitro, molecular targeting to the HER2 receptor was the dominant factor driving cancer cell association. In contrast, size was found to be the key determinant of tumor accumulation in vivo, where molecular targeting increased tumor tissue concentrations for 30 nm but not 100 nm IONP. Similar to the in vitro results, PEGylation did not influence in vivo IONP biodistribution. Thus, the results reported here indicate that the in vitro advantages of molecular targeting may not consistently extend to pre-clinical in vivo settings. These observations may have important implications for the design and clinical translation of advanced, multifunctional, IONP platforms.

DNA Repair Polymorphisms Modify Bladder Cancer Risk: A Multi-factor Analytic Strategy

Objectives: A number of common non-synonymous single nucleotide polymorphisms (SNPs) in DNA repair genes have been reported to modify bladder cancer risk. These include: APE1-Asn148Gln, XRCC1-Arg399Gln and XRCC1-Arg194Trp in the BER pathway, XPD-Gln751Lys in the NER pathway and XRCC3-Thr241Met in the DSB repair pathway. Methods: To examine the independent and interacting effects of these SNPs in a large study group, we analyzed these genotypes in 1,029 cases and 1,281 controls enrolled in two case-control studies of incident bladder cancer, one conducted in New Hampshire, USA and the other in Turin, Italy. Results: The odds ratio among current smokers with the variant XRCC3-241 (TT) genotype was 1.7 (95% CI 1.0–2.7) compared to wild-type. We evaluated gene-environment and gene-gene interactions using four analytic approaches: logistic regression, Multifactor Dimensionality Reduction (MDR), hierarchical interaction graphs, classification and regression trees (CART), and logic regression analyses. All five methods supported a gene-gene interaction between XRCC1-399/XRCC3-241 (p = 0.001) (adjusted OR for XRCC1-399 GG, XRCC3-241 TT vs. wild-type 2.0 (95% CI 1.4–3.0)). Three methods predicted an interaction between XRCC1-399/XPD-751 (p = 0.008) (adjusted OR for XRCC1-399 GA or AA, XRCC3-241 AA vs. wild-type 1.4 (95% CI 1.1–2.0)). Conclusions: These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and highlight the value of using multiple complementary analytic approaches to identify multi-factor interactions.

Spin traps: in vitro toxicity and stability of radical adducts.

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3). The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

Alcohol Retail Density and Demographic Predictors of Health Disparities: A Geographic Analysis

OBJECTIVES We examined whether the geographic density of alcohol retailers was greater in geographic areas with higher levels of demographic characteristics that predict health disparities. METHODS We obtained the locations of all alcohol retailers in the continental United States and created a map depicting alcohol retail outlet density at the US Census tract level. US Census data provided tract-level measures of poverty, education, crowding, and race/ethnicity. We used multiple linear regression to assess relationships between these variables and retail alcohol density. RESULTS In urban areas, retail alcohol density had significant nonlinear relationships with Black race, Latino ethnicity, poverty, and education, with slopes increasing substantially throughout the highest quartile for each predictor. In high-proportion Latino communities, retail alcohol density was twice as high as the median density. Retail alcohol density had little or no relationship with the demographic factors of interest in suburban, large town, or rural census tracts. CONCLUSIONS Greater density of alcohol retailers was associated with higher levels of poverty and with higher proportions of Blacks and Latinos in urban census tracts. These disparities could contribute to higher morbidity in these geographic areas.

Effect on regrowth delay in a murine tumor of scheduling split-dose irradiation based on direct pO2 measurements by electron paramagnetic resonance oximetry

Tumor reoxygenation after irradiation may contribute to a tumors response to subsequent doses of radiation. The timing of reoxygenation in RIF-1 murine tumors was determined using electron paramagnetic resonance (EPR) oximetry with intratumoral implantation of an oxygen-sensitive paramagnetic material (India ink) to monitor the pO2 in individual murine tumors before, during and after three different irradiation schemes. Radiation was given as a single 20-Gy dose or was split into two 10-Gy doses where the second dose of radiation was delivered at the minimum postirradiation tumor pO2 (24-h interval, hypoxic group) or where the second dose of radiation was delivered after reoxygenation had occurred (72-h interval, oxygenated group). The end point for tumor response was time taken to reach double the volume at the time of treatment. There were significantly longer tumor doubling times in the oxygenated compared to the hypoxic group, indicating that the measured changes in pO2 reflected changes in tumor radiosensitivity. A 24-h interval between doses resulted in a delay of reoxygenation in the tumors, while a 72-h interval resulted in a second cycle of hypoxia/reoxygenation. Our results suggest that repeated direct measurements of pO2 in tumors by EPR oximetry could be useful in timing radiation doses to achieve improved local control of tumors.