High-resolution microarray analysis of chromosome 20q in human colon cancer metastasis model systems

Amplification of human chromosome 20q DNA is the most frequently occurring chromosomal abnormality detected in sporadic colorectal carcinomas and shows significant correlation with liver metastases. Through comprehensive high-resolution microarray comparative genomic hybridization and microarray gene expression profiling, we have characterized chromosome 20q amplicon genes associated with human colorectal cancer metastasis in two in vitro metastasis model systems. The results revealed increasing complexity of the 20q genomic profile from the primary tumor-derived cell lines to the lymph node and liver metastasis derived cell lines. Expression analysis of chromosome 20q revealed a subset of over expressed genes residing within the regions of genomic copy number gain in all the tumor cell lines, suggesting these are Chromosome 20q copy number responsive genes. Bases on their preferential expression levels in the model system cell lines and known biological function, four of the over expressed genes mapping to the common intervals of genomic copy gain were considered the most promising candidate colorectal metastasis-associated genes. Validation of genomic copy number and expression array data was carried out on these genes, with one gene, DNMT3B, standing out as expressed at a relatively higher levels in the metastasis-derived cell lines compared with their primary-derived counterparts in both the models systems analyzed. The data provide evidence for the role of chromosome 20q genes with low copy gain and elevated expression in the clonal evolution of metastatic cells and suggests that such genes may serve as early biomarkers of metastatic potential. The data also support the utility of the combined microarray comparative genomic hybridization and expression array analysis for identifying copy number responsive genes in areas of low DNA copy gain in cancer cells. ^

Toxics Release Inventory facilities and childhood cancer: Geographic information systems based approach

Purpose. To examine the association between living in proximity to Toxics Release Inventory (TRI) facilities and the incidence of childhood cancer in the State of Texas. ^ Design. This is a secondary data analysis utilizing the publicly available Toxics release inventory (TRI), maintained by the U.S. Environmental protection agency that lists the facilities that release any of the 650 TRI chemicals. Total childhood cancer cases and childhood cancer rate (age 0-14 years) by county, for the years 1995-2003 were used from the Texas cancer registry, available at the Texas department of State Health Services website. Setting: This study was limited to the children population of the State of Texas. ^ Method. Analysis was done using Stata version 9 and SPSS version 15.0. Satscan was used for geographical spatial clustering of childhood cancer cases based on county centroids using the Poisson clustering algorithm which adjusts for population density. Pictorial maps were created using MapInfo professional version 8.0. ^ Results. One hundred and twenty five counties had no TRI facilities in their region, while 129 facilities had at least one TRI facility. An increasing trend for number of facilities and total disposal was observed except for the highest category based on cancer rate quartiles. Linear regression analysis using log transformation for number of facilities and total disposal in predicting cancer rates was computed, however both these variables were not found to be significant predictors. Seven significant geographical spatial clusters of counties for high childhood cancer rates (p<0.05) were indicated. Binomial logistic regression by categorizing the cancer rate in to two groups (<=150 and >150) indicated an odds ratio of 1.58 (CI 1.127, 2.222) for the natural log of number of facilities. ^ Conclusion. We have used a unique methodology by combining GIS and spatial clustering techniques with existing statistical approaches in examining the association between living in proximity to TRI facilities and the incidence of childhood cancer in the State of Texas. Although a concrete association was not indicated, further studies are required examining specific TRI chemicals. Use of this information can enable the researchers and public to identify potential concerns, gain a better understanding of potential risks, and work with industry and government to reduce toxic chemical use, disposal or other releases and the risks associated with them. TRI data, in conjunction with other information, can be used as a starting point in evaluating exposures and risks. ^

Specialized managed care for children with cystic fibrosis: Resource use patterns and rate design for a high cost Medicaid population

Objective. This research study had two goals: (1) to describe resource consumption patterns for Medi-Cal children with cystic fibrosis, and (2) to explore the feasibility from a rate design perspective of developing specialized managed care plans for such a special needs population.^ Background. Children with special health care needs (CSHN) comprise about 2% of the California Medicaid pediatric population. CSHN have rare but serious health problems, such as cystic fibrosis. Medicaid programs, including Medi-Cal, are enrolling more and more beneficiaries in managed care to control costs. CSHN, however, do not fit the wellness model underlying most managed care plans. Child health advocates believe that both efficiency and quality will suffer if CSHN are removed from regionalized special care centers and scattered among general purpose plans. They believe that CSHN should be "carved out" from enrollment in general plans. One alternative is the Specialized Managed Care Plan, tailored for CSHN.^ Methods. The study population consisted of children under age 21 with CF who were eligible for Medi-Cal and California Children's Services program (CCS) during 1991. Health Care Financing Administration (HCFA) Medicaid Tape-to-Tape data were analyzed as part of a California Children's Hospital Association (CCHA) project.^ Results. Mean Medi-Cal expenditures per month enrolled were $2,302 for 457 CF children, compared to about \$1,270 for all 47,000 CCS special needs children and roughly $60 for almost 2.6 million ``regular needs'' children. For CF children, inpatient care (80\%) and outpatient drugs (9\%) were the major cost drivers, with {\it all\/} outpatient visits comprising only 2\% of expenditures. About one-third of CF children were eligible due to AFDC (Aid to Families with Dependent Children). Age group explained about 17\% of all expenditure variation. Regression analysis was used to select the best capitation rate structure (rate cells by age and eligibility group). Sensitivity analysis estimated moderate financial risk for a statewide plan (360 enrollees), but severe risk for single county implementation due to small numbers of children.^ Conclusions. Study results support the carve out of CSHN due to unique expenditure patterns. The Specialized Managed Care Plan concept appears feasible from a rate design perspective given sufficient enrollees. ^

Design, Calibration, and Evaluation of Depth-of-Interaction-Capable PET Detector Modules

High-resolution, small-bore PET systems suffer from a tradeoff between system sensitivity, and image quality degradation. In these systems long crystals allow mispositioning of the line of response due to parallax error and this mispositioning causes resolution blurring, but long crystals are necessary for high system sensitivity. One means to allow long crystals without introducing parallax errors is to determine the depth of interaction (DOI) of the gamma ray interaction within the detector module. While DOI has been investigated previously, newly available solid state photomultipliers (SSPMs) well-suited to PET applications and allow new modules for investigation. Depth of interaction in full modules is a relatively new field, and so even if high performance DOI capable modules were available, the appropriate means to characterize and calibrate the modules are not. This work presents an investigation of DOI capable arrays and techniques for characterizing and calibrating those modules. The methods introduced here accurately and reliably characterize and calibrate energy, timing, and event interaction positioning. Additionally presented is a characterization of the spatial resolution of DOI capable modules and a measurement of DOI effects for different angles between detector modules. These arrays have been built into a prototype PET system that delivers better than 2.0 mm resolution with a single-sided-stopping-power in excess of 95% for 511 keV g's. The noise properties of SSPMs scale with the active area of the detector face, and so the best signal-to-noise ratio is possible with parallel readout of each SSPM photodetector pixel rather than multiplexing signals together. This work additionally investigates several algorithms for improving timing performance using timing information from multiple SSPM pixels when light is distributed among several photodetectors.

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.