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.

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.

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.

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.

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.