IV (retrospective case series review).

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.

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.

BackgroundRectal 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.MethodsFrom 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.ResultsRespondents 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].ConclusionGiven 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.