Screening for abuse risk in pain patients.

As opioid prescribing has dramatically expanded over the past decade, so too has the problem of prescription drug abuse. In response to these now two major public health problems - the problem of poorly treated chronic pain and the problem of opioid abuse - a new paradigm has arisen in pain management, namely risk stratification. Once a prescriber has determined that opioids will be used (a medical decision based on how intense the pain is, what has been tried and failed and, to some extent, what type of pain the patient has), he/she must then decide how opioid therapy is to be delivered.

A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis.

It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood.

Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment.

There is a strong need for science-based risk assessment that utilizes known data from diverse sources to arrive at accurate assessments of human health risk. Such assessments will protect the public health without mandating unreasonable regulation. This paper utilizes 30 years of research on three "known human carcinogens": formaldehyde, vinyl chloride (VC), and ethylene oxide (EO), each of which forms DNA adducts identical to endogenous DNA adducts in all individuals.

Infrared spatial and frequency selective metamaterial with near-unity absorbance.

We demonstrate, for the first time, a spatially dependent metamaterial perfect absorber operating in the infrared regime. We achieve an experimental absorption of 97% at a wavelength of 6.0   μm, and our results agree well with numerical full-wave simulations.

Substance abuse in cancer pain.

In the oncology community, opioids recently have become the cornerstone of cancer pain management. This has led to a rapid increase in opioid prescribing in an effort to address the growing public health problem of chronic pain. A new paradigm in noncancer pain management has emerged, that of risk assessment and stratification in opioid therapy.

Rab33b and Rab6 are functionally overlapping regulators of Golgi homeostasis and trafficking.

We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra-Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra-Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex- or Zeste White 10 (ZW10)-depletion induced disruption of the Golgi ribbon in HeLa cells.

A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice.

Recent advances in cancer therapeutics stress the need for a better understanding of the molecular mechanisms driving tumor formation. This can be accomplished by obtaining a more complete description of the genes that contribute to cancer. We previously described an approach using the Sleeping Beauty (SB) transposon system to model hematopoietic malignancies in mice.

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium.

A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)-associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC.

Using quantitative fluorescence microscopy to probe organelle assembly and membrane trafficking.

With current light microscopy and laboratory-level computational capability, many questions in organelle assembly and membrane trafficking that were once treated in a qualitative manner can now be treated quantitatively. We present here an overview of the principles involved in doing quantitative fluorescence microscopy. We illustrate these with examples drawn from our work with the Golgi apparatus and endosomes in cultured mammalian cells.

Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse.

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22.

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4(+) T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells.

Supported planar bilayers for study of the immunological synapse.

Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC-peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two-dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid-anchored membrane proteins, 6-histidine-tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers.

Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment.

Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging.

T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation.

Recognition of peptide antigen by T cells involves coordinated movement of T cell receptors (TCRs) along with other costimulatory and signaling molecules. The spatially organized configurations that result are collectively referred to as the immunological synapse. Experimental investigation of the role of spatial organization in TCR signaling has been facilitated by the use of nanopatterned-supported membranes to direct TCR into alternative patterns.

Biomarkers in toxicology and risk assessment: informing critical dose-response relationships.

Tremendous advances have been made in the study of biomarkers related to carcinogenesis during the past 20 years. This perspective will briefly review improvements in methodology and instrumentation that have increased our abilities to measure the formation, repair, and consequences of DNA adducts. These biomarkers of exposure, along with surrogates such as protein adducts, have greatly improved our understanding of species differences in metabolism and effects of chemical stability and DNA repair on tissue differences in molecular dose.

Probabilistic dose-response modeling: case study using dichloromethane PBPK model results.

A revised assessment of dichloromethane (DCM) has recently been reported that examines the influence of human genetic polymorphisms on cancer risks using deterministic PBPK and dose-response modeling in the mouse combined with probabilistic PBPK modeling in humans. This assessment utilized Bayesian techniques to optimize kinetic variables in mice and humans with mean values from posterior distributions used in the deterministic modeling in the mouse. To supplement this research, a case study was undertaken to examine the potential impact of probabilistic rather than deterministic PBPK and dose-response modeling in mice on subsequent unit risk factor (URF) determinations.

Quantification and modeling of tripartite CD2-, CD58FC chimera (alefacept)-, and CD16-mediated cell adhesion.

Alefacept is a chimeric protein combining CD58 immunoglobulin-like domain 1 with human IgG1 Fc. Alefacept mediates adhesion by bridging CD2 on T cells to activating Fc receptors on effector cells, but the equilibrium binding parameters have not been determined. Alefacept mediated T cell killing by NK cells and adhesion between CD2- and CD16-expressing cells at an optimum concentration of 100 nM.

Both post-Golgi and intra-Golgi cycling affect the distribution of the Golgi phosphoprotein GPP130.

Golgi phosphoprotein, GPP130, a cis Golgi protein, is representative of proteins cycling between the Golgi apparatus and endosomes in a pH-sensitive manner. The present qualitative data are insufficient to distinguish the relative contributions of Golgi and endosomal processes in regulating the cycling of such proteins. We have taken a quantitative approach to analyze GPP130 distribution in response to pH perturbation.

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships.

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized.

Ent3p and Ent5p exhibit cargo-specific functions in trafficking proteins between the trans-Golgi network and the endosomes in yeast.

The phosphoinositide-binding proteins Ent3p and Ent5p are required for protein transport from the trans-Golgi network (TGN) to the vacuole in Saccharomyces cerevisiae. Both proteins interact with the monomeric clathrin adaptor Gga2p, but Ent5p also interacts with the clathrin adaptor protein 1 (AP-1) complex, which facilitates retention of proteins such as Chs3p at the TGN. When both ENT3 and ENT5 are mutated, Chs3p is diverted from an intracellular reservoir to the cell surface.

Control of antigen presentation with a photoreleasable agonist peptide.

The immunological synapse is a specialized intercellular junction between a T cell and a target cell that orchestrates the engagement of receptors and ligands in space and time as a means of regulating function. Here we introduce a reagent for controlling the spatial and temporal presentation of natural antigen to T cells. Moth cytochrome c (88-103) peptide (MCC), an agonist to the murine T cell receptor AND when presented in the context of H2 IEk major histocompatibility complex (IEk), was synthesized with the side-chain amine of Lys99 conjugated to a photosensitive protecting group, 6-nitroveratryloxycarbonyl (NVOC).

TCDD exposure-response analysis and risk assessment.

We examined the relation between cancer mortality and time-dependent cumulative exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) estimated from a concentration- and age-dependent kinetic model of elimination, and we estimated incremental cancer risks at age 75. Data from the National Institute for Occupational Safety and Health study of 3,538 workers with occupational exposure to TCDD were analyzed using standardized mortality ratios and Cox regression procedures. Analyses adjusted for potential confounding by age, year of birth, and race and considered exposure lag periods of 0, 10, or 15 years.

Workshop overview: reassessment of the cancer risk of dichloromethane in humans.

The U.S. Environmental Protection Agency (U.