Surface-Induced Hydrophobin Assemblies with Versatile Properties and Distinct Underlying Structures.

Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EASΔ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues.

Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function.

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones; some sHsp family members are upregulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation; however, fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a single-molecule fluorescence-based approach to observe the chaperone action of human alphaB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1. By examining the dispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation.

Sexual HIV risk among gay, bisexual and queer transgender men: findings from interviews in Vancouver, Canada.

Gay, bisexual, queer and other men who have sex with men are disproportionately affected by HIV in Canada. While up to 63% of transgender men identify as gay, bisexual or queer and report a variety of HIV sexual risk behaviours, transgender men are often overlooked within epidemiological HIV surveillance and research. While a growing body of research has begun to examine sexual risk for transgender gay, bisexual and queer men, most studies have been conducted in the USA. This study explored sexual HIV risk for this population in the Canadian context, specifically in British Columbia, in an environment of publically funded universal access to healthcare, including HIV testing and treatment. We conducted interviews with 11 gay, bisexual and queer transgender men. Participants' narratives suggest that HIV risk for these transgender men is shaped by a diversity of sexual behaviours, including inconsistent condom use, seeking partners online for greater safety and accessing HIV/STI testing and other healthcare services despite facing transition-related barriers. Public health prevention and health education must recognise the presence of transgender men and ensure health services and broader population health promotion meet the unique sexual health needs of this sub-population of gay, bisexual and queer men.

Substance use patterns and unprotected sex among street-involved youth in a Canadian setting: a prospective cohort study.

BACKGROUND: Rates of sexually transmitted infections (STI) and unplanned pregnancy are high among youth. While the intersection between drug and alcohol use and unprotected sex is well recognized, few studies have examined the relationship between substance use patterns and unprotected sex among high risk-populations such as street-involved youth. METHODS: Data were derived from the At-Risk Youth Study (ARYS), a prospective cohort of street-involved youth from Vancouver, Canada. Generalized estimating equations (GEE) were used to examine substance use patterns that were independently associated with unprotected sex, defined as (vaginal or anal) sexual intercourse without consistent condom use. RESULTS: Between September 2005 and May 2013, 1,026 youth were recruited into the ARYS cohort and 75% (n = 766) reported engaging in recent unprotected sex at some point during the study period. In a multivariable analysis, female gender (adjusted odds ratio [AOR] = 1.46, 95% confidence interval [CI]: 1.18-1.81), Caucasian ancestry (AOR = 1.38, 95% CI: 1.13-1.68), being in a stable relationship (AOR = 4.64, 95% CI: 3.82-5.65), having multiple sex partners (AOR = 2.60, 95% CI: 2.18-3.10) and the following substance use patterns were all independently associated with recent unprotected sex: injection or non-injection crystal methamphetamine use (AOR = 1.21, 95% CI: 1.03-1.43), injection or non-injection cocaine use (AOR = 1.20, 95% CI: 1.02-1.41), marijuana use (AOR = 1.23, 95% CI: 1.02-1.49), ecstasy use (AOR = 1.23, 95% CI: 1.01-1.48) and alcohol use (AOR = 1.31, 95% CI: 1.11-1.55) (all p < 0.05). CONCLUSIONS: Unprotected sex was prevalent among street-involved youth in this setting, and independently associated with female gender and a wide range of substance use patterns. Evidence-based and gender-informed sexual health interventions are needed in addition to increased access to youth-centered addiction treatment services. STI testing and linkages to healthcare professionals remain important priorities for street-involved youth, and should be integrated across all health and social services.

Community-based HIV and STI prevention in women working in indoor sex markets.

Community research into women's experiences in the indoor commercial sex industry illustrated an urgent need for sexually transmitted infection (STI) and HIV education, prevention, testing, and treatment and culturally appropriate services to support the sexual and reproductive health of commercial sex workers (CSWs). This work also revealed that a high number of immigrant--primarily Asian--women are involved in the indoor sex industry. In response, the authors developed a community-academic research partnership to design and implement a blended outreach research program to provide STI and HIV prevention interventions for indoor CSWs and their clients. This Community Health Worker Model HIV Prevention and Health Promotion Program incorporated health education, primary care referrals, STI testing using self-swab techniques, and a point-of-care HIV screening test. Here the authors report on program implementation, design, and the experiences of participants and team members and provide research and vaccination recommendations for future work in this area. This work work affirms that community-based service providers can be a key entry point for indoor CSWs to access health care and sexual health promotion and education and may be a solution to missed opportunities to provide culturally and contextually appropriate education and services to this population.

Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality.

Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-ß scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric ß-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.

Violence in the massage parlor industry: experiences of Canadian-born and immigrant women.

We examined and contrasted 129 Canadian-born and immigrant women's experiences of violence and associated structural and interpersonal factors within indoor commercial sex venues. The majority experienced at least one form of structural, interpersonal, or both types of violence, with the attempted removal of a condom during sexual services being cited most frequently. Canadian-born women reported more frequent violent assaults in the survey data. The women's qualitative narratives illustrated that perceptions of violence differed significantly among Canadian versus non-Canadian born women. Findings concerning racialization and gendered relations of power have important implications for prevention and interventions to support victims of abuse.

Structure and agency: reflections from an exploratory study of Vancouver indoor sex workers.

Sex work research continues to be characterised by debates around decriminalization. Central to these debates are claims about the agency of those involved in the sex trade. Some researchers argue that individuals involved in the sex trade are victims of structural and interpersonal constraint, whilst others depict them as workers exercising choice. Drawing on structure-agency theory, a review of legal and media accounts of the sex trade and qualitative interviews with 21 indoor sex workers in Vancouver, Canada, we argue that both of these perspectives are insufficient. Rather than reducing the sex trade to part of a binary, we suggest that it is necessary to analyse sex work through the complex interplay of both structure and agency. Specifically, structural analyses undercover the numerous ways that sex workers are controlled, observed and influenced whilst agency perspectives elicit the means that sex workers continue to exercise control in spite of disadvantage. While we do not finalise decriminalisation debates, we do critique current Canadian laws for the lack of responsiveness to the lives of sex workers and their exploitative and contradictory stance on sex work.

An α-Cyanostilbene Derivative for the Enhanced Detection and Imaging of Amyloid Fibril Aggregates.

The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Benzothiazole dyes such as Thioflavin T (ThT) are well-characterized and widely used fluorescent probes for monitoring amyloid fibril formation. However, existing dyes lack sensitivity and specificity to oligomeric intermediates formed during fibril formation. In this work, we describe the use of an α-cyanostilbene derivative (called ASCP) with aggregation-induced emission properties as a fluorescent probe for the detection of amyloid fibrils. Similar to ThT, ASCP is fluorogenic in the presence of amyloid fibrils and, upon binding and excitation at 460 nm, produces a red-shifted emission with a large Stokes shift of 145 nm. ASCP has a higher binding affinity to fibrillar α-synuclein than ThT and likely shares the same binding sites to amyloid fibrils. Importantly, ASCP was found to also be fluorogenic in the presence of amorphous aggregates and can detect oligomeric species formed early during aggregation. Moreover, ASCP can be used to visualize fibrils via total internal reflection fluorescence microscopy and, due to its large Stokes shift, simultaneously monitor the fluorescence emission of other labelled proteins following excitation with the same laser used to excite ASCP. Consequently, ASCP possesses enhanced and unique spectral characteristics compared to ThT that make it a promising alternative for the in vitro study of amyloid fibrils and the mechanisms by which they form.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

Characteristics of injection drug users who participate in drug dealing: implications for drug policy.

So-called "balanced" drug policy couples enforcement initiatives targeting drug dealers with health-focused interventions serving addicted individuals. There are few evaluations of this approach, and little is known about how these two populations may overlap. We evaluated factors associated with drug dealing among injection drug users (IDUs) in Vancouver, Canada, and examined self-reported drug-dealing roles and reasons for dealing. Among 412 IDUs seen from March through December 2005, 68 (17%) had dealt drugs during the previous six months. Variables independently associated with drug dealing included: recent incarceration (adjusted odds ratio [AOR] = 2.9; 95%CI: 1.4-6.0); frequent heroin injection (AOR = 2.5; 95%CI: 1.4-4.6); frequent cocaine injection (AOR = 2.0; 95%CI: 1.1-3.8); and recent overdose (AOR = 2.7; 95%CI: 1.0-7.3). The most common drug-dealing roles were direct selling (82%), middling (35%), and steering (19%), while the most common reasons for dealing included obtaining drugs (49%) and money (36%). Drug dealing among IDUs was predicted by several markers of higher intensity addiction, and drug-dealing IDUs tended to occupy the most dangerous positions in the drug-dealing hierarchy. These findings suggest that elements of "balanced" drug policies may undermine each other and indicate the need for alternative interventions.

Exploring Health Service Underutilization: A Process Evaluation of the Newcomer Women's Health Clinic.

The BC Women's Hospital and Health Centre's Newcomer Women's Health Clinic (NWHC) opened in 2014 to fill a gap in services for newcomer women to Canada residing in Metro Vancouver. After 1 year the NWHC was operating at only 50% capacity. An evaluation was conducted to better understand the underutilization of clinic services. Evaluation data was collected via patient intake forms, patient feedback surveys, and one-to-one interviews with both newcomer women and service providers who work with newcomer women. Results highlight the heterogeneity in newcomer women's experiences including different perceptions of health, health needs, and health-seeking behaviours. Despite the population's diversity, some common experiences exist, including difficulties navigating and accessing a new health system and lack of awareness of the NWHC. Findings provide insight for improving the delivery of NWHC services, and contribute to a rich and ongoing dialogue concerning the diversity of experiences of newcomer women in Canada.

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function.

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.

Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits.

Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs) that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP), with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.

Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor.

Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse.

Sexual violence among a cohort of injection drug users.

The objective of this study was to determine the prevalence of, and factors associated with, sexual violence in childhood, adolescence and adulthood, among injection drug using men and women. The Vancouver Injection Drug User Study is a prospective cohort of injection drug users (IDU) begun in 1996. The analysis included all individuals who completed the baseline questionnaire who responded to a question about sexual assault. Multivariate modeling was used to determine and to what extent a history of sexual violence at different ages is predictive of HIV risk and other health risk behaviors. HIV prevalence was calculated as the total current number of HIV-positive individuals in the cohort. Of the 1437 eligible individuals, 36% reported a lifetime history of sexual violence; 68% of women, and 19% of men (p<0.001). After adjusting for fixed sociodemographics, these individuals were more likely to have ever been in the sex trade, to knowingly share needles/rigs with HIV-positive people, to have attempted suicide, to have ever accidentally overdosed, to binge on alcohol, and to have been diagnosed with a mental disorder/disability. The prevalence of child sexual abuse in this cohort is 21%; 33% for women, and 13% for men. The data show a dose-response relationship between age at first sexual violence and most risk behaviors examined. These relationships are further mediated by gender. The prevalence of HIV among individuals who ever experienced sexual violence was 25%, compared to 19% among those who never experienced sexual violence (p=0.006). Sexual violence, and especially child sexual abuse, is highly prevalent among this cohort, particularly among women. Child sexual abuse has worse consequences for both genders than sexual violence later in life. Nevertheless, women and men are affected differently by sexual violence at different ages, and this has significant implications for health promotion programs, and specifically HIV prevention.

Offer of financial incentives for unprotected sex in the context of sex work.

INTRODUCTION AND AIMS: Commercial sex workers (CSW) are often portrayed as vectors of disease transmission. However, the role clients play in sexual risk taking and related decision making has not been thoroughly characterised. DESIGN AND METHODS: Participants were drawn from the Vancouver Injection Drug Users Study, a longitudinal cohort. Analyses were restricted to those who reported selling sex between June 2001 and December 2005. Using multivariate generalised estimating equation, we evaluated the prevalence of and factors associated with being offered money for sex without a condom. RESULTS: A total of 232 CSW were included in the analyses, with 73.7% reporting being offered more money for condom non-use, and 30.6% of these CSW accepting. Variables independently associated with being offered money for sex without a condom included daily speedball use [adjusted odds ratio (AOR) = 1.21, 95% confidence interval (CI): 0.23-0.62], daily crack smoking (AOR = 1.51, 95% CI: 1.04-2.19), daily heroin injection (AOR = 1.76, 95% CI: 1.27-2.43) and drug use with clients (AOR = 3.22, 95% CI: 2.37-4.37). Human immunodeficiency virus seropositivity was not significant (AOR = 0.98, 95% CI: 0.67-1.44). DISCUSSION AND CONCLUSIONS: Findings highlight the role clients play in contributing to unprotected sex through economic influence and exploitation of CSW drug use. HIV serostatus has no bearing on whether more money is offered for sex without a condom. Novel interventions should target both CSW and clients.

Clients of indoor commercial sex workers: heterogeneity in patronage patterns and implications for HIV and STI propagation through sexual networks.

OBJECTIVES: To determine whether "high-risk" clients occupied important sociometric positions in sexual networks of commercial sex workers and to estimate whether they were more likely to be HIV and STI infected. GOAL: To determine whether a classification of high-risk clients could be validated by network analysis. STUDY DESIGN: We used proxy data on clients collected from a cross-sectional survey of 49 indoor female sex workers. RESULTS: Two types of clients were categorized as high risk, including those who created sexual bridges between sex establishments and those who had sex with most or all the FSW at an establishment. High-risk clients were significantly more central and were more likely to be members of cohesive subgroups than were lower-risk clients. The few known HIV and STI infections were in high-risk clients. CONCLUSIONS: It is possible to identify theoretically high-risk commercial sex clients from the network perspective using simple data collection and categorization approaches.

Conducting HIV/AIDS research with indoor commercial sex workers: reaching a hidden population.

BACKGROUND: Although comprising up to 80% of the commercial sex industry in Canada, indoor female sex workers (FSW) are generally not represented in research because they are a hidden population and difficult to access. OBJECTIVES: This paper describes a community-academic partnership model that was established to gain access to, deliver outreach services to, and conduct community-based research with the indoor commercial sex industry in four cities in British Columbia. METHODS: The project employed an ongoing community consultation, peer-delivered approach to reaching this overlooked segment of the commercial sex industry. Peers (former and current FSW) were hired, trained, and supported as outreach workers and participated in the development, implementation, and evaluation of the project. Outreach teams visited sex establishments to deliver harm reduction materials and provide education, support, and referrals. The teams developed rapport with establishment managers and staff to facilitate research recruitment and data collection. The community team leader met with managers in targeted business to describe the study and elicit permission to recruit workers. The team leader conducted in-person interviews with consenting FSW. OBSERVATIONS: During the first 2 years of the project, more than 50 sex establishments were visited by outreach teams and 37 allowed repeat visits. Research interviews have been conducted with 49 FSW in seven establishments from four cities. CONCLUSIONS: Although the high cost in terms of time and resources must be recognized, this project represents a successful research and outreach model that permits access to the hidden commercial sex industry.