Optimising and personalising behavioural healthcare in the US Department of Defense through Primary Care Behavioral Health.

Over the past 25 years, one way the US Department of Defense (DoD) has worked to optimise and personalise the delivery of behavioural healthcare is by integrating behavioural health providers into primary care settings. Using the Primary Care Behavioral Health (PCBH) model for integration allows behavioural health providers to see service members and their families for brief and targeted appointments. These appointments are focused on ensuring that the patient receives the care that is needed, while reducing the barriers (eg, delays in receiving care, negative stigma, isolated from other medical care) that are often associated with seeking behavioural healthcare. We review the primary components of the PCBH model, detail the history of how the DoD implemented the PCBH model, review the training methods used by the DoD and briefly describe some of the research that has been conducted by the DoD evaluating the PCBH model.

RNAi and iTRAQ reagents united: targeted quantitation of siRNA-mediated protein silencing in human cells.

Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

A sensitive fluorescence monitor for the detection of activated Ras: total chemical synthesis of site-specifically labeled Ras binding domain of c-Raf1 immobilized on a surface.

BACKGROUND: The Ras.GDP-Ras.GTP cycle plays a central role in eukaryotic signaling cascades. Mutations in Ras which stabilize activated Ras.GTP lead to a continuous stimulation of downstream effectors and ultimately to cell proliferation. Ras mutants which increase the steady-state concentration of Ras.GTP are involved in about 30% of all human cancers. It is therefore of great interest to develop a biosensor which is sensitive to Ras.GTP but not to Ras.GDP. RESULTS: The Ras binding domain (RBD) of c-Raf1 was synthesized from two unprotected peptide segments by native chemical ligation. Two fluorescent amino acids with structures based on the nitrobenz-2-oxa-1,3-diazole and coumaryl chromophores were incorporated at a site which is close to the RBD/Ras.GTP binding surface. Additionally, a C-terminal tag consisting of His6 was introduced. The Kd values for binding of the site-specifically modified proteins to Ras.GTP are comparable to that of wild-type RBD. Immobilization of C-terminal His6 tag-modified fluorescent RBD onto Ni-NTA-coated surfaces allowed the detection of Ras.GTP in the 100 nM range. Likewise, Ras.GTP/Q61L (an oncogenic mutant of Ras with very low intrinsic GTP hydrolysis activity) can also be detected in this assay system. Ras.GDP does not bind to the immobilized RBD, thus allowing discrimination between inactive and activated Ras. CONCLUSIONS: The site-specific incorporation of a fluorescent group at a strategic position in a Ras effector protein allows the detection of activated Ras with high sensitivity. This example illustrates the fact that the chemical synthesis of proteins or protein domains makes it possible to incorporate any kind of natural or unnatural amino acid at the position of choice, thereby enabling the facile preparation of specific biosensors, enhanced detection systems for drug screening, or the synthesis of activated proteins, e.g. phosphorylated proteins involved in signaling pathways, as defined molecular species.

Demographic, lifestyle, and psychosocial predictors of frequency of intoxication and other indicators as estimates of alcohol-related problems in Air Force basic military recruits.

This study examined demographic, lifestyle, and psychosocial variables to determine predictors of Air Force recruits who are likely to have alcohol-related problems. Subjects were all Air Force recruits (N = 32,144) entering basic training from August 1995 to September 1996. The dependent measures were self-reported frequency of eight or more drinks per occasion, frequency of fighting while drinking, and typical frequency of alcohol consumption. Demographic analysis revealed that individuals high on any dependent variable were more likely to be male, older, non-Hispanic whites with some college. Lifestyle predictors included positive attitudes toward drug use and smoking status, with risk greater for females than males and for non-whites than non-Hispanic whites at the same smoking level. Psychosocial predictors included positive rebellious attitudes, decreased seatbelt use, and positive risk-taking attitudes, with risk greater for females than males at the same risk attitude level. These findings suggest that problem drinking falls into a broader category of risky problem behaviors.

Structural and functional definition of the human chitinase chitin-binding domain.

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, beta-1, 3-glucan, beta-1,3-1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding to Saccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.

Role of the heme propionates in the interaction of heme with apomyoglobin and apocytochrome b5.

The heme propionate groups of both myoglobin (Mb) and cytochrome b5 form hydrogen bonds with nearby surface amino acids residues that are believed to stabilize the heme-protein complex. To evaluate the magnitude of this stabilization, the kinetics of heme dissociation from variants of horse heart Mb and cytochrome b5 in which these hydrogen bonding interactions have been systematically eliminated were studied by the method of Hargrove and colleagues (1994), and their thermal stability was assessed. Elimination of each hydrogen bond was found to decrease the thermal stability of the proteins and increase the rate constant for heme dissociation in a progressive fashion. For the Mb derivatives, 1H-NMR studies indicate that the elimination of individual hydrogen bonds also affects the rate at which the heme orientational equilibrium is achieved. In both types of kinetics experiment, the effects of decreasing the number of potential hydrogen bonding interactions are found to be cumulative. Despite their kinetic effects, elimination of these hydrogen bonding interactions had no influence on the initial distribution of heme orientational isomers immediately following reconstitution or on the equilibrium constant of heme orientational disorder. The interactions between the heme propionates and nearby protein residues play a partial role in the stabilization of the heme-protein complex and are a major factor in the kinetic "trapping" of the minor heme orientation. Comparisons of the various rate constants determined for the mechanism of heme binding and reorientation suggests that the intramolecular reorientation mechanism is slightly favored over the intermolecular mechanism.

Dissociation of heme from myoglobin and cytochrome b5: comparison of behavior in solution and the gas phase.

The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme-protein complexes in the orifice-skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabilized in solution but stabilized in the gas-phase ions. These results suggest that on the millisecond time scale of the ES-MS experiment, the gas-phase protein ion may retain much of the structure of the protein in solution, at least for those residues surrounding the heme group. Furthermore, the ability of ES-MS to detect relatively subtle differences in protein-small molecule complex stability demonstrated in this work suggests that this technique may be a convenient, sensitive, and generally useful strategy for physical characterization of such complexes.

FTIR analysis of the interaction of azide with horse heart myoglobin variants.

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile variants is accompanied by a shift of the nu max for the low-spin infrared band to lower energy and by a significant increase in the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histidine and bound azide, and in the other conformation no interaction occurs.

Methanesulfonyl fluoride enhances one-trial reward learning in mid-aged rats.

In previous studies, 18-month-old rats have shown no significant retention 24 hours after the single acquisition trial in a one-trial discriminative reward learning task. In the present study, ten 18-month-old rats pretreated with 0.5 mg/kg MSF IP showed significantly better retention in terms of speed and errors than eleven 18-month-old rats pretreated with injection vehicle alone. However, twelve two-three-month-old rats pretreated with the same dose of MSF failed to show better retention than eleven two-three-month-old rats pretreated with vehicle alone.