Cross-Validation of the JSORRAT-II in Iowa.

The predictive validity of the Juvenile Sexual Offense Recidivism Risk Assessment Tool-II (JSORRAT-II) was evaluated using an exhaustive sample of 11- to 17-year-old male juveniles who offended sexually (JSOs) between 2000 and 2006 in Iowa (n = 529). The validity of the tool in predicting juvenile sexual recidivism was significant (area under the receiver operating characteristic curve [AUC] = .70, 99% confidence interval [CI] = [.60, .81], d = 0.70). Non-significant predictive validity coefficients were observed for the prediction of non-sexual forms of recidivism. Additional analyses were undertaken to test hypotheses about the tool's performance with various subsamples. The age of the JSO at the time of the index sexual offense and time at risk outside secure facility placements interacted significantly with JSORRAT-II scores to predict juvenile sexual recidivism. The implications of these findings for practice and research on the validation of risk assessment tools are discussed.

Dietary estimates of dioxins consumed in U.S. Department of Agriculture-regulated meat and poultry products.

The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) examined whether levels of dioxin-like compounds (DLCs) measured in FSIS-regulated meat and poultry products indicate possible concern for U.S. public health based on usual and recommended consumption patterns of meat and poultry for the U.S. population. The FSIS estimated daily dietary exposures and compared them with the reference dose (RfD) established by the U.S. Environmental Protection Agency (EPA) for potential noncancer risks from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), assuming that all measured DLCs were represented by the RfD (i.e., not just TCDD alone). The estimates indicate that a typical U.S. adult daily exposure of DLCs from FSIS-regulated products is below the EPA-established RfD. Only children consuming chronic average daily servings of meat or poultry products containing the highest measured levels of DLCs may exceed the RfD. If one follows the recommendations from the 2010 Dietary Guidelines for Americans, all expected exposures to DLCs from FSIS-regulated products are estimated to be well below the RfD.

Aß alters the connectivity of olfactory neurons in the absence of amyloid plaques in vivo.

The amyloid beta peptide aggregates into amyloid plaques at presymptomatic stages of Alzheimer's disease, but the temporal relationship between plaque formation and neuronal dysfunction is poorly understood. Here we demonstrate that the connectivity of the peripheral olfactory neural circuit is perturbed in mice overexpressing human APPsw (Swedish mutation) before the onset of plaques. Expression of human APPsw exclusively in olfactory sensory neurons also perturbs connectivity with associated reductions in odour-evoked gene expression and olfactory acuity. By contrast, olfactory sensory neuron axons project correctly in mice overexpressing wild-type human amyloid precursor protein throughout the brain and in mice overexpressing M671V human APP, a missense mutation that reduces amyloid beta production, exclusively in olfactory sensory neurons. Furthermore, expression of Aß40 or Aß42 solely in the olfactory epithelium disrupts the olfactory sensory neuron axon targeting. Our data indicate that altering the structural connectivity and function of highly plastic neural circuits is one of the pleiotropic actions of soluble human amyloid beta.

Evaluation of FKBP and DHFR based destabilizing domains in Saccharomyces cerevisiae.

Two orthogonal destabilizing domains have been developed based on mutants of human FKBP12 as well as bacterial DHFR and these engineered domains have been used to control protein concentration in a variety of contexts in vitro and in vivo. FKBP12 based destabilizing domains cannot be rescued in the yeast Saccharomyces cerevisiae; ecDHFR based destabilizing domains are not degraded as efficiently in S. cerevisiae as in mammalian cells or Plasmodium, but provide a starting point for the development of domains with increased signal-to-noise in S. cerevisiae.

"Boys don't cry": examination of the links between endorsement of masculine norms, self-stigma, and help-seeking attitudes for men from diverse backgrounds.

The role of conformity to dominant U.S. masculine norms as an antecedent to help-seeking attitudes in men has been established using convenience samples made up largely of college-age and European American males. However, the role of conformity to masculine norms on help-seeking attitudes for noncollege-age men or for men from diverse backgrounds is not well understood. To fill this gap in the literature, the present study examined the cross-cultural relevance of a mediational model of the relationships between conformity to dominant U.S. masculine norms and attitudes toward counseling through the mediator of self-stigma of seeking counseling for 4,773 men from both majority and nonmajority populations (race/ethnicity and sexual orientation). Structural equation modeling results showed that the model established using college males from majority groups (European American, heterosexual) may be applicable to a community sample of males from differing racial/ethnic groups and sexual orientations. However, some important differences in the presence and strengths of the relationships between conformity to dominant masculine norms and the other variables in the model were present across different racial/ethnic groups and sexual orientations. These findings suggest the need to pay specific theoretical and clinical attention to how conformity to dominant masculine norms and self-stigma are linked to unfavorable attitudes toward help seeking for these men, in order to encourage underserved men's help-seeking behavior.

Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae.

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co-expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5'-gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast-enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast-enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon-optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest.

The rapamycin-binding domain of the protein kinase mammalian target of rapamycin is a destabilizing domain.

Rapamycin is an immunosuppressive drug that binds simultaneously to the 12-kDa FK506- and rapamycin-binding protein (FKBP12, or FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) kinase. The resulting ternary complex has been used to conditionally perturb protein function, and one such method involves perturbation of a protein of interest through its mislocalization. We synthesized two rapamycin derivatives that possess large substituents at the C-16 position within the FRB-binding interface, and these derivatives were screened against a library of FRB mutants using a three-hybrid assay in Saccharomyces cerevisiae. Several FRB mutants responded to one of the rapamycin derivatives, and twenty of these mutants were further characterized in mammalian cells. The mutants most responsive to the ligand were fused to yellow fluorescent protein, and fluorescence levels in the presence and absence of the ligand were measured to determine stability of the fusion proteins. Wild-type and mutant FRB domains were expressed at low levels in the absence of the rapamycin derivative, and expression levels rose up to 10-fold upon treatment with ligand. The synthetic rapamycin derivatives were further analyzed using quantitative mass spectrometry, and one of the compounds was found to contain contaminating rapamycin. Furthermore, uncontaminated analogs retained the ability to inhibit mTOR, although with diminished potency relative to rapamycin. The ligand-dependent stability displayed by wild-type FRB and FRB mutants as well as the inhibitory potential and purity of the rapamycin derivatives should be considered as potentially confounding experimental variables when using these systems.

pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15).

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.