Bioimaging with Macromolecular Probes Incorporating Multiple BODIPY Fluorophores.

Seven macromolecular constructs incorporating multiple borondipyrromethene (BODIPY) fluorophores along a common poly(methacrylate) backbone with decyl and oligo(ethylene glycol) side chains were synthesized. The hydrophilic oligo(ethylene glycol) components impose solubility in aqueous environment on the overall assembly. The hydrophobic decyl chains effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. As a result, the brightness of these multicomponent assemblies is approximately three times greater than that of a model BODIPY monomer. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. The covalent scaffold of these macromolecular constructs also permits their subsequent conjugation to secondary antibodies. The covalent attachment of polymer and biomolecule does not hinder the targeting ability of the latter and the resulting bioconjugates can be exploited to stain the tubulin structure of model cells to enable their visualization with optimal signal-to-noise ratios. These results demonstrate that this particular structural design for the incorporation of multiple chromophores within the same covalent construct is a viable one to preserve the photophysical properties of the emissive species and enable the assembly of bioimaging probes with enhanced brightness.

Fluorescent Kinase Probes Enabling Identification and Dynamic Imaging of HER2(+) Cells.

The human epidermal growth factor receptor, EGFR/ERBB/HER, family of receptor tyrosine kinases is central to many signaling pathways and a validated chemotherapy target in multiple cancers. While EGFR/ERBB-targeted therapies, including monoclonal antibodies, e.g., trastuzumab, and small molecule kinase inhibitors, such as lapatinib, have been developed, rapid identification and classification of cancer cells is key to identifying the best treatment regime. We report ERBB2 (also HER2) targeting kinase probes that exhibit a "turn-on" emission response upon binding. These live cell compatible probes differentiate ERBB2(+) cells from low-level, ERBB2(-) cells by targeting the intracellular ATP-binding pocket of ERBB2 with therapeutic inhibitor-like specificity. Beyond kinase expression levels, probe signal is linked to the phosphotyrosine-correlated activation state of the ERBB2 population. Additionally, the rapid signaling capability of the probes can report changes in activation state in live cells providing a unique type of complementary information to immunohistochemical assays of receptor kinase populations.

Binding-induced, turn-on fluorescence of the EGFR/ERBB kinase inhibitor, lapatinib.

We report the photophysical properties, binding-induced turn-on emission, and fluorescence imaging of the cellular uptake and distribution of lapatinib, an EGFR/ERBB inhibitor. Lapatinib, a type II, i.e. inactive state, inhibitor that targets the ATP binding pocket of the EGFR family of receptor tyrosine kinases. DFT calculations predict that the 6-furanylquinazoline core of lapatinib should exhibit an excited state with charge transfer character and an S0 to S1 transition energy of 3.4 eV. Absorption confirms an optical transition in the near UV to violet, while fluorescence spectroscopy shows that photoemission is highly sensitive to solvent polarity. The hydrophobicity of lapatinib leads to fluorescent aggregates in solution, however, binding to the lipid-carrier protein, BSA or to the kinase domain of ERBB2, produces spectroscopically distinct photoemission. Confocal fluorescence microscopy imaging of lapatinib uptake in ERBB2-overexpressing MCF7 and BT474 cells reveals pools of intracellular inhibitor with emission profiles consistent with aggregated lapatinib.

One probe, two-channel imaging of nuclear and cytosolic compartments with orange and red emissive dyes.

Several new DNA-targeting probes that exhibit binding-induced 'turn on' fluorescence are presented. Two of the dyes, orange emissive 1, (E)-4-(4(-4-methylpiperazin-1-yl)phenyl)6-(4-(4-methylpi-perazin-1-yl)styryl)pyrimidin-2-ol), and red emissive 2, (E)-4-(4(-4-methyl-piperazin-1-yl)-phenyl)6-(4-(4-methylpiperazin-1-yl)styryl)-1,3-propanedionato-κO,κO']difluoroborane), are brightly fluorescent when bound to DNA, but are virtually non-fluorescent in aqueous solutions. Confocal fluorescence microscopy of live BT474, MCF7 and HEK293 cells demonstrates that both probes are cell permeable and rapidly accumulated intracellularly into cell nuclei and the cytosol. Taking advantage of their environmental sensitivity, these two pools of fluorophores are readily resolved into separate channels, and thus, a single dye allows two-color imaging of the nuclear and cytosolic compartments.

Impact of Individual Budgets on Work and Financial Well-Being of Supported Employment Recipients With Serious Mental Illness.

OBJECTIVE: The authors sought to determine whether providing recipients of supported employment with individual budgets from which they could purchase employment-related goods and services would improve employment and financial outcomes. METHODS: Sixty study participants were recruited from an individual placement and support (IPS) program and randomly assigned (1:1) to receive IPS services only (N=32) or IPS services with a 12-month $950 flexible fund called a career account (N=28). Participants receiving IPS and a career account met with staff who helped them identify employment goals and create a budget for purchases directly tied to these goals. The primary outcome was competitive employment; secondary outcomes included job tenure, days worked, total earnings, and financial well-being. Outcomes were analyzed by using adjusted generalized linear models (GLMs) with binary logistic, negative binomial, and linear distributions. RESULTS: The proportion of participants who achieved competitive employment was largely similar for those in the career account+IPS group (54%) and in the IPS-only group (47%). However, the GLM analysis revealed that career account+IPS participants had significantly longer job tenure, more total days of employment, and higher total earnings than IPS-only participants. Feelings of financial well-being increased significantly among career account participants, whereas financial well-being declined among control participants. The amount of career account dollars participants spent was positively and significantly associated with longer job tenure, more days employed, and higher total earnings. CONCLUSIONS: Combining flexible funds with IPS-supported employment achieved some superior outcomes compared with IPS only. Further research is needed to assess the longer-term effects of this practice and its cost-effectiveness.

A Mixed-Methods Implementation Evaluation of Virtual Reality Job Interview Training in IPS Supported Employment.

OBJECTIVE: Employment rates among individuals with serious mental illness may be improved by engagement in the individual placement and support (IPS) model of supported employment. Results from a recent randomized controlled trial (RCT) indicate that virtual reality job interview training (VR-JIT) improves employment rates among individuals with serious mental illness who have been actively engaged in IPS for at least 90 days. This study reports on an initial implementation evaluation of VR-JIT during the RCT in a community mental health agency. METHODS: A sequential, complementary mixed-methods design included use of qualitative data to improve understanding of quantitative findings. Thirteen IPS staff trained to lead VR-JIT implementation completed VR-JIT acceptability, appropriateness, and feasibility surveys. Participants randomly assigned to IPS with VR-JIT completed acceptability (N=42) and usability (N=28) surveys after implementation. The authors also conducted five focus groups with IPS staff (N=11) and VR-JIT recipients (N=13) and semistructured interviews with IPS staff (N=9) and VR-JIT recipients (N=4), followed by an integrated analysis process. RESULTS: Quantitative results suggest that IPS staff found VR-JIT to be highly acceptable, appropriate for integration with IPS, and feasible for delivery. VR-JIT was highly acceptable to recipients. Qualitative results add important context to the quantitative findings, including benefits of VR-JIT for IPS staff as well as adaptations for delivering technology-based interventions to individuals with serious mental illness. CONCLUSIONS: These qualitative and quantitative findings are consistent with each other and were influenced by VR-JIT's adaptability and perceived benefits. Tailoring VR-JIT instruction and delivery to individuals with serious mental illness may help optimize VR-JIT implementation within IPS.

Psychometric properties of the mock interview rating scale for schizophrenia and other serious mental illnesses.

Background: Over the past 10 years, job interview training has emerged as an area of study among adults with schizophrenia and other serious mental illnesses who face significant challenges when navigating job interviews. The field of mental health services research has limited access to assessments of job interview skills with rigorously evaluated psychometric properties. Objective: We sought to evaluate the initial psychometric properties of a measure assessing job interview skills via role-play performance. Methods: As part of a randomized controlled trial, 90 adults with schizophrenia or other serious mental illnesses completed a job interview role-play assessment with eight items (and scored using anchors) called the mock interview rating scale (MIRS). A classical test theory analysis was conducted including confirmatory factor analyses, Rasch model analysis and calibration, and differential item functioning; along with inter-rater, internal consistency, and test-retest reliabilities. Pearson correlations were used to evaluate construct, convergent, divergent, criterion, and predictive validity by correlating the MIRS with demographic, clinical, cognitive, work history measures, and employment outcomes. Results: Our analyses resulted in the removal of a single item (sounding honest) and yielded a unidimensional total score measurement with support for its inter-rater reliability, internal consistency, and test-retest reliability. There was initial support for the construct, convergent, criterion, and predictive validities of the MIRS, as it correlated with measures of social competence, neurocognition, valuing job interview training, and employment outcomes. Meanwhile, the lack of correlations with race, physical health, and substance abuse lent support for divergent validity. Conclusion: This study presents initial evidence that the seven-item version of the MIRS has acceptable psychometric properties supporting its use to assess job interview skills reliably and validly among adults with schizophrenia and other serious mental illnesses. Clinical Trial Registration: NCT03049813.

Probing the functional limits of the norepinephrine transporter with self-reporting, fluorescent stilbazolium dimers.

A series of stilbazolium dimers were synthesized and investigated as sterically demanding ligands targeting the norepinephrine transporter (NET). The environmentally sensitive fluorescence of the dyes enables their use as self-reporting ligands; binding to and displacement from NET can be monitored by fluorescence microscopy.

Fluorescent stilbazolium dyes as probes of the norepinephrine transporter: structural insights into substrate binding.

We report the synthesis, binding kinetics, optical spectroscopy and predicted binding modes of a series of sterically demanding, fluorescent norepinephrine transporter (NET) ligands. A series of bulky stilbazolium dyes, including six newly synthesized compounds, were evaluated to determine the effect of extending the molecular probes' 'heads' or 'tails'. Taking advantage of the dyes' characteristic 'turn-on' emission, the kinetic binding parameters, k(on) and k(off) were determined revealing that extension of the molecules' tails is well tolerated while expansion of the head is not. Additionally, a 'headfirst' orientation appears to be preferred over a 'tail-first' binding pose. Further details of the possible binding modes were obtained from the emission spectra of the bound probes. A small range of interplanar twist angles, approximately 35° to 60°, is predicted to produce the observed emission. Docking experiments and molecular modelling support the kinetic and spectroscopic data providing structural insights into substrate binding.

An RCT of Virtual Reality Job Interview Training for Individuals With Serious Mental Illness in IPS Supported Employment.

OBJECTIVE: Virtual Reality Job Interview Training (VR-JIT) is a computerized interview simulator with efficacy at enhancing interview skills and employment outcomes. A randomized controlled trial assessed VR-JIT effectiveness for participants in individual placement and support (IPS), in which approximately 55% of individuals with serious mental illness obtain employment. METHODS: Ninety participants with serious mental illness were randomly assigned to IPS+VR-JIT (N=54) or IPS as usual (N=36), completing pretest-posttest assessments and an employment evaluation at 9 months. Intent-to-treat chi-square analysis, multivariable logistic regression, Cox proportional hazards models, and mixed-effects linear regressions were conducted. Fifty-one percent were IPS nonresponders (i.e., no employment within the first 90 days of IPS). RESULTS: IPS+VR-JIT participants did not have significantly higher employment rates, compared with IPS-as-usual participants (43% versus 28%). IPS nonresponders (N=46) in the IPS+VR-JIT group had greater odds of obtaining employment (odds ratio [OR]=5.82, p=0.014) and shorter time to employment (hazard ratio=2.70, p=0.044) compared with IPS nonresponders in the IPS-as-usual group. Intent-to-treat mixed-effects linear analyses indicated that IPS+VR-JIT, compared with IPS as usual, significantly improved interview skills (p=0.006), interview confidence (p=0.013), and interview anxiety (p=0.019). CONCLUSIONS: VR-JIT's potential benefits (increased employment in a shorter time) appeared to be specific to IPS nonresponders, whereas employment outcomes for recent IPS enrollees were not affected. VR-JIT could be a valuable resource for employment specialists to support IPS nonresponders, because 47% of participants engaged in mock interview training with their specialist. Future research should focus on evaluating the effectiveness and implementation of VR-JIT among IPS nonresponders.

Luminescent charge-transfer complexes: tuning emission in binary fluorophore mixtures.

Charge-transfer (CT) complexes composed of a π-electron-poor naphthalene diimide (NDI) derivative combined with a series of π-electron-rich donors were investigated. Solutions of the CT complexes are nonemissive; however, solid-state complexes and aqueous suspensions display emission that is dependent on the energy of the HOMO of the electron donor. Crystallographic analysis of a pyrene-NDI complex reveals columnar packing and a high degree of frontier molecular orbital (FMO) overlap that likely contributes to the observed optical properties. The fluorescent CT particles are utilized as imaging agents; additional luminescent CT complexes may be realized by considering FMO energies and topologies.

Fluorescent reporters of monoamine transporter distribution and function.

Serotonin is a monoamine serving as a chemical messenger in diverse brain regions, as well as in blood and various other organs. We synthesized six ethylamine functionalized fluorophores as fluorescent probes for serotonin. The one with best spectral properties and aqueous solubility, 6-amino-2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione, was studied in detail both in vivo and in vitro. It was shown to act as a ligand for serotonin transporter (SERT) without acute cerebral or cardiovascular toxicity or adverse effects. Fluorescent serotonin analogs can be used for direct visualization of SERT distribution and activity in live tissue.

Fluorescent neuroactive probes based on stilbazolium dyes.

A set of spectrally diverse stilbazolium dyes was identified in an uptake assay using cultured brainstem and cerebellum cells isolated from e19 chicks. Pretreatment of cells with indatraline, a monoamine reuptake inhibitor, allowed identification of dyes that may interact with monoamine transporters. Two structurally related, yet spectrally segregated, probes, (E)-1-methyl-4-[2-(2-naphthalenyl)ethenyl]-pyridinium iodide (NEP+, 3A) and (E)-4-[2-(6-hydroxy-2-naphthalenyl)ethenyl]-1-methyl-pyridinium iodide (HNEP+, 4A), were selected and further investigated using HEK-293 cells selectively expressing dopamine, norepinephrine or serotonin transporters. HNEP+ was selectively accumulated via catecholamine transporters, with the norepinephrine transporter (NET) giving the highest response; NEP+ was not transported, though possible binding was observed. The alternate modes of interaction enable the use of NEP+ and HNEP+ to image distinct cell populations in live brain tissue explants. The preference for HNEP+ accumulation via NET was confirmed by imaging uptake in the absence and presence of desipramine, a norepinephrine reuptake inhibitor.

Psychosocial risk factors for inconsistent condom use in young people with first episode psychosis.

There is evidence of high rates of unprotected sex among young people with first episode psychosis compared to their peers. Little research has explored factors associated with condom use in this population. The current study examined the association between previously identified psychosocial risk factors and condom use in young people with early psychosis and their peers. Sixty-seven sexually active young people with first episode psychosis and 48 sexually active control participants matched on a number of sociodemographic factors completed a self-report survey. Increased probability of inconsistent condom use was associated with clinical status, younger age, unemployment, and the absence of peer support for condom use. Psychological distress, self-esteem, social support, substance use, and impulsivity were not associated with condom use. The results suggest that sexual risk-reduction interventions for young people with psychosis should target peer norms, particularly among those who are younger and unemployed.

Stress induces a switch of intracellular signaling in sensory neurons in a model of generalized pain.

Stress dramatically exacerbates pain in diseases such as fibromyalgia and rheumatoid arthritis, but the underlying mechanisms are unknown. We tested the hypothesis that stress causes generalized hyperalgesia by enhancing pronociceptive effects of immune mediators. Rats exposed to nonhabituating sound stress exhibited no change in mechanical nociceptive threshold, but showed a marked increase in hyperalgesia evoked by local injections of prostaglandin E(2) or epinephrine. This enhancement, which developed more than a week after exposure to stress, required concerted action of glucocorticoids and catecholamines at receptors located in the periphery on sensory afferents. The altered response to pronociceptive mediators involved a switch in coupling of their receptors from predominantly stimulatory to inhibitory G-proteins (G(s) to G(i)), and for prostaglandin E(2), emergence of novel dependence on protein kinase C epsilon. Thus, an important mechanism in generalized pain syndromes may be stress-induced coactivation of the hypothalamo-pituitary-adrenal and sympathoadrenal axes, causing a long-lasting alteration in intracellular signaling pathways, enabling normally innocuous levels of immune mediators to produce chronic hyperalgesia.

Sexual dimorphism in the effect of sound stress on neutrophil function.

It has been hypothesized that stress contributes to the severity of inflammatory diseases. However, the mechanisms underlying this effect are incompletely understood. In this study we investigated the effects of sound stress on function of the polymorphonuclear neutrophil-immune cells that play key roles in both the acute and chronic inflammatory response. Specifically, we examined the effect of stress on the production of reactive oxygen species (ROS) and phagocytosis by rat neutrophils, and the role of sympathoadrenal stress axis in these effects. Since many inflammatory diseases exhibit sexual dimorphism, we also investigated the contribution of sex and gonadal hormones to the effects of stress on neutrophil function. Peripheral blood neutrophils were harvested from male and female rats exposed to intermittent sound stress (over 4 days). Stress suppressed ROS production in males (but not females) an effect that was eliminated in adrenal medullectomized males. Stress also suppressed neutrophil phagocytosis in males and females. Again, this effect was absent following adrenal medullectomy. To investigate the role of sex hormones in these sexual dimorphic responses to stress, rats were gonadectomized prepubertally and exposed to stress as adults. In gonadectomized males, stress produced an even larger decrease in ROS production, but had no effect on the stress-induced inhibition of phagocytosis. Gonadectomy prevented the stress-induced inhibition of neutrophil phagocytosis in females. These data indicate that the adrenal medulla, perhaps via release of epinephrine, suppresses neutrophil ROS production in males and phagocytosis in males and females.

STIs and blood borne viruses - risk factors for individuals with mental illness.

BACKGROUND: Individuals with mental illness have poorer sexual health compared to the general population and experience a number of barriers to accessing health care. This population demonstrates elevated rates of sexually transmitted infections (STIs) and blood borne viruses (BBVs), as well as higher rates of associated risky behaviours. OBJECTIVE: To review factors associated with increased risk for STI/BBV infection among individuals with mental health problems, and to discuss the potential implications for general practice management. DISCUSSION: There are multiple factors associated with risk for STI/BBV infection among people with mental health problems. An awareness of relevant risk factors is necessary for providing a timely and comprehensive approach. Relevant strategies include sexual health and STI/BBV screening, education/counselling, diagnosis, treatment, skills training and appropriate referral. General practitioners play an important role in ensuring the sexual health needs of those with mental heath issues are adequately addressed.

beta(2)-Adrenergic receptor-dependent sexual dimorphism for murine leukocyte migration.

In wild-type FVB mice, leukocyte recruitment to lipopolysaccharide was sexually dimorphic, with a greater number of leukocytes recruited in females. In male beta(2)-adrenergic receptor knock out mice (bred on a congenic FVB background) the number of leukocytes recruited was increased approximately 4-fold, while in females there was no change, eliminating sexual dimorphism in leukocyte migration. While there were significantly fewer recruited CD62L(+) and CD11a(+) leukocytes in wild-type males, only in male beta-adrenergic receptor knock out mice was there an increase in the number of recruited CD11a(+) leukocytes, again eliminating sexual dimorphism. Thus, leukocyte migration and CD11a(+) adhesion molecule expression in male, but not in female, leukocytes is beta-adrenergic receptor-dependent. Our findings provide support for a role of beta(2)-adrenergic receptor mechanisms in the inflammatory response, and suggest that beta(2)-adrenergic receptor on male leukocytes contributes to sexual dimorphism in the effect of stress on inflammatory diseases.

Oat beta-glucan effects on neutrophil respiratory burst activity following exercise.

UNLABELLED: Fatiguing exercise has been associated with a decrease in certain functions of neutrophils, whereas moderate exercise has generally been associated with an increase. Consumption of oat beta-glucan (ObetaG), a soluble fiber and mild immune system enhancer, may offset the immunosuppression associated with intense training and perhaps further enhance the benefits of moderate exercise. PURPOSE: To test the effects of ObetaG consumption on neutrophil function and number after both moderate and fatiguing exercise. METHODS: Male mice were assigned to one of six treatment groups. Fatiguing exercise mice (Ftg-ObetaG and Ftg-H2O) ran to volitional fatigue on a treadmill for three consecutive days, and moderate exercise mice (Mod-ObetaG and Mod-H2O) ran for six consecutive days for 1 h. Control mice (Con-ObetaG and Con-H2O) were exposed to the treadmill environment but did not run. ObetaG was consumed in the drinking water (approximately 0.6 mL x d(-1)) for 10 consecutive days. After rest or exercise on the last day of training, mice were given a 1-mL i.p. injection of thioglycollate. Mice were sacrificed 3 h later; neutrophils were harvested from the peritoneal cavity and counted, and their respiratory burst activity was measured using flow cytometry. RESULTS: Both moderate exercise and ObetaG increased neutrophil burst activity, whereas fatiguing exercise had no effect. Neutrophil number was increased by fatiguing exercise and ObetaG, but not moderate exercise. There were no additive effects of exercise and ObetaG on either of these variables. CONCLUSION: These data suggest that although not additive in their effects, both ObetaG and exercise can alter overall neutrophil respiratory burst activity (number and/or function), but only ObetaG increased both number and function, which may have important ramifications for defense against infection.

Highlighting Cancer Cells with Halochromic Switches.

Halochromic coumarin-oxazine prefluorophores and targeting folate ligands can be connected covalently to the side chains of amphiphilic polymers. The resulting macromolecular constructs assemble into nanoparticles in aqueous environments. The prefluorophores do not produce any detectable fluorescence at neutral pH, but are converted into fluorophores with intense visible emission at acidic pH. Protonation opens the oxazine heterocycle to shift bathochromically the coumarin absorption and activate fluorescence with a brightness per nanoparticle approaching 5 × 105 M-1 cm-1. This value translates into a 170-fold enhancement relative to the isolated fluorophores dissolved in organic solvent. The folate ligands direct these multicomponent constructs into acidic intracellular compartments of folate-positive cells, where the prefluorophores switch to the corresponding fluorophores and produce fluorescence. The pH-induced activation of the signaling units ensures negligible background fluorescence from the extracellular matrix, which instead limits considerably the contrast accessible with model systems incorporating conventional nonactivatable fluorophores. Furthermore, no intracellular fluorescence can be detected when the very same measurements are performed with folate-negative cells. Nonetheless, control experiments demonstrate that the covalent connection of the prefluorophores to the polymer backbone of the amphiphilic constructs is essential to ensure selectivity. Model systems with prefluorophores noncovalently encapsulated cannot discriminate folate-positive from -negative cells. Thus, our structural design for the covalent integration of activatable signaling units and targeting ligands within the same nanostructured assembly together with the photophysical properties engineered into the emissive components offer the opportunity to highlight cancer cells selectively with high brightness and optimal contrast.