Modulation of a protein-folding landscape revealed by AFM-based force spectroscopy notwithstanding instrumental limitations.

Single-molecule force spectroscopy is a powerful tool for studying protein folding. Over the last decade, a key question has emerged: how are changes in intrinsic biomolecular dynamics altered by attachment to µm-scale force probes via flexible linkers? Here, we studied the folding/unfolding of α3D using atomic force microscopy (AFM)-based force spectroscopy. α3D offers an unusual opportunity as a prior single-molecule fluorescence resonance energy transfer (smFRET) study showed α3D's configurational diffusion constant within the context of Kramers theory varies with pH. The resulting pH dependence provides a test for AFM-based force spectroscopy's ability to track intrinsic changes in protein folding dynamics. Experimentally, however, α3D is challenging. It unfolds at low force (<15 pN) and exhibits fast-folding kinetics. We therefore used focused ion beam-modified cantilevers that combine exceptional force precision, stability, and temporal resolution to detect state occupancies as brief as 1 ms. Notably, equilibrium and nonequilibrium force spectroscopy data recapitulated the pH dependence measured using smFRET, despite differences in destabilization mechanism. We reconstructed a one-dimensional free-energy landscape from dynamic data via an inverse Weierstrass transform. At both neutral and low pH, the resulting constant-force landscapes showed minimal differences (∼0.2 to 0.5 kBT) in transition state height. These landscapes were essentially equal to the predicted entropic barrier and symmetric. In contrast, force-dependent rates showed that the distance to the unfolding transition state increased as pH decreased and thereby contributed to the accelerated kinetics at low pH. More broadly, this precise characterization of a fast-folding, mechanically labile protein enables future AFM-based studies of subtle transitions in mechanoresponsive proteins.

Type III secretion system effector proteins are mechanically labile.

Multiple gram-negative bacteria encode type III secretion systems (T3SS) that allow them to inject effector proteins directly into host cells to facilitate colonization. To be secreted, effector proteins must be at least partially unfolded to pass through the narrow needle-like channel (diameter <2 nm) of the T3SS. Fusion of effector proteins to tightly packed proteins-such as GFP, ubiquitin, or dihydrofolate reductase (DHFR)-impairs secretion and results in obstruction of the T3SS. Prior observation that unfolding can become rate-limiting for secretion has led to the model that T3SS effector proteins have low thermodynamic stability, facilitating their secretion. Here, we first show that the unfolding free energy ([Formula: see text]) of two Salmonella effector proteins, SptP and SopE2, are 6.9 and 6.0 kcal/mol, respectively, typical for globular proteins and similar to published [Formula: see text] for GFP, ubiquitin, and DHFR. Next, we mechanically unfolded individual SptP and SopE2 molecules by atomic force microscopy (AFM)-based force spectroscopy. SptP and SopE2 unfolded at low force (Funfold ≤ 17 pN at 100 nm/s), making them among the most mechanically labile proteins studied to date by AFM. Moreover, their mechanical compliance is large, as measured by the distance to the transition state (Δx‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast, prior measurements of GFP, ubiquitin, and DHFR show them to be mechanically robust (Funfold > 80 pN) and brittle (Δx‡ < 0.4 nm). These results suggest that effector protein unfolding by T3SS is a mechanical process and that mechanical lability facilitates efficient effector protein secretion.

Is HIV prevention creating new biosocialities among gay men? Treatment as prevention and pre-exposure prophylaxis in Canada.

The advancements of "treatment as prevention" (TasP), "undetectable viral load" (UVL) and "pre-exposure prophylaxis" (PrEP) are redefining HIV prevention standards. Relying on the concept of biosociality, this article explores how gay men rally around, debate, and sometimes disagree about these emerging HIV prevention technologies. This article is based on data from the Resonance Project, a Canadian community-based research project. Twelve focus groups (totalling 86 gay and bisexual men) were held in three Canadian cities (Montreal, Toronto, Vancouver) in 2013-2014. Respondents view UVL and PrEP through the prism of their generational experience of HIV prevention. In this respect, biosocialities highlight an experiential dimension that is tied to the context of the HIV epidemic. The biosocialities of HIV prevention are also built around serological identities. However, our study shows the diversity of these positions. Analysis grounded in biosocialities is useful for better understanding how scientific information circulates, is made sense of, and generates debate among gay men.

Force Spectroscopy with 9-µs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 µm2 cantilever into one terminated with a gold-coated, 4 × 4 µm2 reflective region connected to an uncoated 2-µm-wide central shaft. This "Warhammer" geometry achieved 8.5-µs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein. Additionally, nonspecific surface adhesion hinders studies of α-helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet-Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Optimizing 1-µs-Resolution Single-Molecule Force Spectroscopy on a Commercial Atomic Force Microscope.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is widely used to mechanically measure the folding and unfolding of proteins. However, the temporal resolution of a standard commercial cantilever is 50-1000 µs, masking rapid transitions and short-lived intermediates. Recently, SMFS with 0.7-µs temporal resolution was achieved using an ultrashort (L = 9 µm) cantilever on a custom-built, high-speed AFM. By micromachining such cantilevers with a focused ion beam, we optimized them for SMFS rather than tapping-mode imaging. To enhance usability and throughput, we detected the modified cantilevers on a commercial AFM retrofitted with a detection laser system featuring a 3-µm circular spot size. Moreover, individual cantilevers were reused over multiple days. The improved capabilities of the modified cantilevers for SMFS were showcased by unfolding a polyprotein, a popular biophysical assay. Specifically, these cantilevers maintained a 1-µs response time while eliminating cantilever ringing (Q ≅ 0.5). We therefore expect such cantilevers, along with the instrumentational improvements to detect them on a commercial AFM, to accelerate high-precision AFM-based SMFS studies.

Prevalence and types of rectal douches used for anal intercourse: results from an international survey.

BACKGROUND: Rectal products used with anal intercourse (AI) may facilitate transmission of STIs/HIV. However, there is limited data on rectal douching behavior in populations practicing AI. We examined the content, types of products, rectal douching practices and risk behaviors among those reporting AI. METHODS: From August 2011 to May 2012, 1,725 women and men reporting receptive AI in the past 3 months completed an internet-based survey on rectal douching practices. The survey was available in English, French, German, Mandarin, Portuguese, Russian, Spanish, and Thai and included questions on sexual behaviors associated with AI including rectal douching. Differences by rectal douching practices were evaluated using chi-square methods and associations between reported douching practices and other factors including age and reported STI history were evaluated using logistic regression analysis. RESULTS: Respondents represented 112 countries, were mostly male (88%), and from North America (55%) or Europe (22%). Among the 1,339 respondents (66%) who reported rectal douching, most (83%) reported always/almost always douching before receptive AI. The majority of rectal douchers reported using non-commercial/homemade products (93%), with water being the most commonly used product (82%). Commercial products were used by 31%, with the most common product being saline-based (56%). Rectal douching varied by demographic and risk behaviors. The prevalence of rectal douching was higher among men (70% vs. 32%; p-value < .01), those reporting substance-use with sex (74% vs. 46%; p-value < .01), and those reporting an STI in the past year (69% vs. 57% p-value < .01) or ever testing HIV-positive (72% vs. 53%; p-value < .01). In multivariable analysis, adjusting for age, gender, region, condom and lubricant use, substance use, and HIV-status, douchers had a 74% increased odds of reporting STI in the past year as compared to non-douchers [adjusted odds ratio (AOR) = 1.74; 95% CI 1.01-3.00]. CONCLUSION: Given that rectal douching before receptive AI is common and because rectal douching was associated with other sexual risk behaviors the contribution of this practice to the transmission and acquisition of STIs including HIV may be important.

Wired Sex Assemblages Among Men Who Have Sex with Men: Sexualized Drug Use, Hookup Apps, and HIV Service Provision.

Sexualized drug use is a form of sexual practice that resists risk-based discourses (otherwise referred to as "radical sex practices") and is reportedly common among gay, bisexual and other men who have sex with men (GBMSM). With the growth of online technologies, the use of hookup apps has also increased. We refer to men's use of drugs, apps, and sex form as "wired sex" that forms what post-structuralist theorists Deleuze and Guattari described as an assemblage. Perspectives of the health and social service providers who work directly with GBMSM has not been explored. This research project involved a critical discourse analysis of 13 semi-structured interviews with service providers in Canada to understand their perspectives and interactions with wired sex assemblages. We identified several themes reflecting the social and political effects of wired sex assemblages and discuss the implications of these effects on services provision with GBMSM.

Risk of sexual transmission of HIV in the context of viral load suppression.

Background: In 2018, the Public Health Agency of Canada (PHAC) published a systematic review to calculate the risk of sexual transmission of human immunodeficiency virus (HIV) in the context of antiretroviral therapy (ART). In 2022, PHAC commissioned the Canadian Agency for Drugs and Technologies in Health (CADTH) to conduct a rapid review of evidence published since 2017. We undertook a meta-analysis of relevant studies from these two reviews. Methods: Studies from the rapid review that adequately assessed exposure (HIV viral load) and outcome (HIV seroconversion) were included and assessed for risk of bias (RoB) and certainty of evidence. Results were pooled to estimate the risk of HIV transmission per 100 person-years. Results: Three studies from the rapid review were eligible for inclusion and one was excluded after RoB assessment. In the remaining studies examining risk among people living with HIV who take ART and maintain a suppressed viral load (fewer than 200 copies/mL, measured every 4-6 months), no sexual transmissions of HIV were observed. The pooled incidence estimate based on these studies, and one from the 2018 PHAC review, was zero transmissions/100 person-years (95% CI: 0.00-0.10). No studies in the rapid review provided data on the risk of sexual transmission of HIV in situations of varying levels of viral load. Conclusion: This update highlights the consistency of evidence since the 2018 PHAC review. There remains no evidence of HIV transmission to sexual partners when a person living with HIV is on ART and maintains a suppressed viral load.

Anatomic Versus Mechanically Aligned Total Knee Arthroplasty for Unicompartmental Knee Arthroplasty Revision.

OBJECTIVES: The purpose of this study was to compare the intra-operative benefits and the clinical outcomes from kinematic or mechanical alignment for total knee arthroplasty (TKA) in patients undergoing revision of failed unicompartmental kneel arthroplasty (UKA) to TKA. METHODS: Ten revisions were performed with a kinematic alignment technique and 11 with a mechanical alignment. Measurements of the hip-knee-ankle angle (HKA), the lateral distal femoral angle (LDFA), and the medial proximal tibial angle (MPTA) were performed using long-leg radiographs. The need for augments, stems, and constrained inserts was compared between groups. Clinical outcomes were compared using the WOMAC score along with maximum distance walked as well as knee range of motion obtained prior to discharge. All data was obtained by a retrospective review of patient files. RESULTS: The kinematic group required less augments, stems, and constrained inserts than the mechanical group and thinner polyethylene bearings. There were significant differences in the lateral distal femoral angle (LDFA) and the medial proximal tibial angle (MPTA) between the two groups (p<0.05). The mean WOMAC score obtained at discharge was better in the kinematic group as was mean knee flexion. At last follow up of 34 months for the kinematic group and 58 months for the mechanical group, no orthopedic complications or reoperations were recorded. CONCLUSION: Although this study has a small patient cohort, our results suggest that kinematic alignment for TKA after UKA revision is an attractive method. Further studies are warranted.

Is wetter better? An evaluation of over-the-counter personal lubricants for safety and anti-HIV-1 activity.

Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. Therefore, safety and anti-HIV-1 activity of over-the-counter (OTC) aqueous- (n = 10), lipid- (n = 2), and silicone-based (n = 2) products were tested. The rheological properties of the lipid-based lubricants precluded testing with the exception of explant safety testing. Six aqueous-based gels were hyperosmolar, two were nearly iso-osmolar, and two were hypo-osmolar. Evaluation of the panel of products showed Gynol II (a spermicidal gel containing 2% nonoxynol-9), KY Jelly, and Replens were toxic to Lactobacillus. Two nearly iso-osmolar aqueous- and both silicone-based gels were not toxic toward epithelial cell lines or ectocervical or colorectal explant tissues. Hyperosmolar lubricants demonstrated reduction of tissue viability and epithelial fracture/sloughing while the nearly iso-osmolar and silicon-based lubricants showed no significant changes in tissue viability or epithelial modifications. While most of the lubricants had no measurable anti-HIV-1 activity, three lubricants which retained cell viability did demonstrate modest anti-HIV-1 activity in vitro. To determine if this would result in protection of mucosal tissue or conversely determine if the epithelial damage associated with the hyperosmolar lubricants increased HIV-1 infection ex vivo, ectocervical tissue was exposed to selected lubricants and then challenged with HIV-1. None of the lubricants that had a moderate to high therapeutic index protected the mucosal tissue. These results show hyperosmolar lubricant gels were associated with cellular toxicity and epithelial damage while showing no anti-viral activity. The two iso-osmolar lubricants, Good Clean Love and PRÉ, and both silicone-based lubricants, Female Condom 2 lubricant and Wet Platinum, were the safest in our testing algorithm.

Coordination Chemistry of Polyaromatic Thiosemicarbazones 2: Synthesis and Biological Activity of Zinc, Cobalt, and Copper Complexes of 1-(Naphthalene-2-yl)ethanone Thiosemicarbazone.

A novel thiosemicarbazone from 2-acetonaphthone (represented as acnTSC) has been synthesized and its basic coordination chemistry with zinc(II), cobalt(II), and copper(II) explored. The complexes were characterized by elemental analysis and various spectroscopic techniques and are best formulated as [M(acnTSC)(2)Cl(2)] with the metal likely in an octahedral environment. The anticancer activity of the complexes was determined against a panel of human colon cancer cells (HCT-116 and Caco-2). The compounds bind to DNA via an intercalative mode with binding constants of 9.7 × 10(4) M(-1), 1.8 × 10(5) M(-1), and 9.5 × 10(4) M(-1) for the zinc, cobalt, and copper complexes, respectively.

Preference and practices relating to lubricant use during anal intercourse: implications for rectal microbicides.

BACKGROUND: The importance of the acceptability of rectal microbicides for HIV and sexually transmissible infections (STIs) prevention is widely recognised. Given relatively consistent use of lubricants for anal intercourse (AI) and the potential for lubricant-like rectal microbicides, understanding barriers to lubricant use may help inform hurdles likely to be encountered once a rectal microbicide becomes available. METHODS: We conducted an internet-based survey using a 25-item questionnaire to assess AI and lubricant use, including lubricant preferences and barriers to use. RESULTS: The majority of the 6124 respondents who reported AI were male (93%), 25 years or older (80%) and from North America (70%). Consistent condom use during AI was reported by a minority (35%) and consistent lubricant use was reported by over half of respondents. Reasons for non-use differed by age and region. Among men, those <25 years were more likely to report barriers around cost compared with those 45 and older (odds ratio (OR) = 6.64; 95% confidence interval (CI) 3.14-14.03). European men (OR = 1.92; 95% CI 1.50-2.45), Latin American women (OR = 3.69; 95% CI 1.27-10.75) and Asian women (OR = 4.04; 95% CI 1.39-11.78) were more likely to report sexual preference as a reason for non-use. CONCLUSIONS: Rectal lubricants are widely used, but barriers to use vary by age and region for dry sex. A lubricant-like rectal microbicide would potentially be acceptable and such a product may be useful as a method of HIV prevention. However, targeted marketing and educational approaches may be needed to enhance use and acceptability of such a product.