4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Magic-angle-spinning NMR structure of the kinesin-1 motor domain assembled with microtubules reveals the elusive neck linker orientation.

Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility, and cell division. Two motors, kinesin and cytoplasmic dynein link the MT network to transported cargos using ATP for force generation. Here, we report an all-atom NMR structure of nucleotide-free kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure reveals the position and orientation of the functionally important neck linker and how ADP induces structural and dynamic changes that ensue in the neck linker. These results demonstrate that the neck linker is in the undocked conformation and oriented in the direction opposite to the KIF5B movement. Chemical shift perturbations and intensity changes indicate that a significant portion of ADP-KIF5B is in the neck linker docked state. This study also highlights the unique capability of MAS NMR to provide atomic-level information on dynamic regions of biological assemblies.

Electronic modulation of metal-support interactions improves polypropylene hydrogenolysis over ruthenium catalysts.

Ruthenium (Ru) is the one of the most promising catalysts for polyolefin hydrogenolysis. Its performance varies widely with the support, but the reasons remain unknown. Here, we introduce a simple synthetic strategy (using ammonia as a modulator) to tune metal-support interactions and apply it to Ru deposited on titania (TiO2). We demonstrate that combining deuterium nuclear magnetic resonance spectroscopy with temperature variation and density functional theory can reveal the complex nature, binding strength, and H amount. H2 activation occurs heterolytically, leading to a hydride on Ru, an H+ on the nearest oxygen, and a partially positively charged Ru. This leads to partial reduction of TiO2 and high coverages of H for spillover, showcasing a threefold increase in hydrogenolysis rates. This result points to the key role of the surface hydrogen coverage in improving hydrogenolysis catalyst performance.

A systematic review and meta-analysis of the evidence for community-based HIV testing on men's engagement in the HIV care cascade.

OBJECTIVE: Men with HIV are less likely than women to know their status, be on antiretroviral therapy, and be virally suppressed. This review examined men's community-based HIV testing services (CB-HTS) outcomes. DESIGN: Systematic review and meta-analysis. METHODS: We searched seven databases and conference abstracts through July 2018. We estimated pooled proportions and/or risk ratios (for meta-analyses) for each outcome using random effects models. RESULTS: 188 studies met inclusion criteria. Common testing models included targeted outreach (e.g. mobile testing), home-based testing, and testing at stand-alone community sites. Across 25 studies reporting uptake, 81% (CI: 75-86%) of men offered testing accepted it. Uptake was higher among men reached through CB-HTS than facility-based HTS (RR = 1.39; CI: 1.13-1.71). Over 69% (CI: 64-71%) of those tested through CB-HTS were men, across 184 studies. Across studies reporting new HIV-positivity among men (n = 18), 96% were newly diagnosed (CI: 77-100%). Across studies reporting linkage to HIV care (n = 8), 70% (CI: 36-103%) of men were linked to care. Across 57 studies reporting sex-disaggregated data for CB-HTS conducted among key populations, men's uptake was high (80%; CI: 70-88%) and nearly all were newly diagnosed and linked to care (95%; CI: 94-100%; and 94%; CI: 88-100%, respectively). CONCLUSION: CB-HTS is an important strategy for reaching undiagnosed men with HIV from the general population and key population groups, particularly using targeted outreach models. When compared to facility-based HIV testing services, men tested through CB-HTS are more likely to uptake testing, and nearly all men who tested positive through CB-HTS were newly diagnosed. Linkage to care may be a challenge following CB-HTS, and greater efforts and research are needed to effectively implement testing strategies that facilitate rapid ART initiation and linkage to prevention services.

Use and awareness of and willingness to self-test for HIV: an analysis of cross-sectional population-based surveys in Malawi and Zimbabwe.

BACKGROUND: Many southern African countries are nearing the global goal of diagnosing 90% of people with HIV by 2020. In 2016, 84 and 86% of people with HIV knew their status in Malawi and Zimbabwe, respectively. However, gaps remain, particularly among men. We investigated awareness and use of, and willingness to self-test for HIV and explored sociodemographic associations before large-scale implementation. METHODS: We pooled responses from two of the first cross-sectional Demographic and Health Surveys to include HIV self-testing (HIVST) questions in Malawi and Zimbabwe in 2015-16. We investigated sociodemographic factors and sexual risk behaviours associated with previously testing for HIV, and past use, awareness of, and future willingness to self-test using univariable and multivariable logistic regression, adjusting for the sample design and limiting analysis to participants with a completed questionnaire and valid HIV test result. We restricted analysis of willingness to self-test to Zimbabwean men, as women and Malawians were not systematically asked this question. RESULTS: Of 31,385 individuals, 31.2% of men had never tested compared with 16.5% of women (p < 0.001). For men, the likelihood of having ever tested increased with age. Past use and awareness of HIVST was very low, 1.2 and 12.6%, respectively. Awareness was lower among women than men (9.1% vs 15.3%, adjusted odds ratio [aOR] = 1.55; 95% confidence interval [CI]: 1.37-1.75), and at younger ages, and lower education and literacy levels. Willingness to self-test among Zimbabwean men was high (84.5%), with greater willingness associated with having previously tested for HIV, being at high sexual risk (highest willingness [aOR = 3.74; 95%CI: 1.39-10.03, p < 0.009]), and being ≥25 years old. Wealthier men had greater awareness of HIVST than poorer men (p < 0.001). The highest willingness to self-test (aOR = 3.74; 95%CI: 1.39-10.03, p < 0.009) was among men at high HIV-related sexual risk. CONCLUSIONS: In 2015-16, many Malawian and Zimbabwean men had never tested for HIV. Despite low awareness and minimal HIVST experience, willingness to self-test was high among Zimbabwean men, especially older men with moderate-to-high HIV-related sexual risk. These data provide a valuable baseline against which to investigate population-level uptake of HIVST as programmes scale up. Programmes introducing, or planning to introduce, HIVST should consider including relevant questions in population-based surveys.

High-resolution 13 C and 43 Ca solid-state NMR and computational studies of the ethylene glycol solvate of atorvastatin calcium.

We report 43 Ca and 13 C solid-state nuclear magnetic resonance (NMR) spectroscopic studies of the ethylene glycol solvate of atorvastatin calcium. The 13 C and 43 Ca chemical shift and 43 Ca quadrupolar coupling tensor parameters are reported. The results are interpreted in terms of the reported X-ray diffraction crystal structure of the solvate and are compared with the NMR parameters of atorvastatin calcium trihydrate, the active pharmaceutical ingredient in Lipitor®. Hartree-Fock and density functional theory calculations of the NMR parameters based on a cluster model derived from the optimized X-ray diffraction crystal structure of the ethylene glycol solvate of atorvastatin calcium are in reasonable agreement with the experimental 43 Ca and 13 C NMR measurables.

Who are the missing men? Characterising men who never tested for HIV from population-based surveys in six sub-Saharan African countries.

INTRODUCTION: We sought to characterize men who had never tested for HIV, understand factors associated with not testing, and measure survey HIV test uptake among never testers. We analysed nationally representative Demographic and Health Surveys of six African countries from 2013 to 2016: Ethiopia, Malawi, Zimbabwe, Rwanda, Lesotho and Zambia. METHODS: Eligible men were household residents or overnight visitors aged 15 to 59 years. We analysed questionnaire responses on HIV testing, known behavioural risk factors, and corresponding HIV laboratory results. We used survey-weighted logistic regression to identify factors associated with never testing for HIV. RESULTS: Approximately double the proportion of men had never tested for HIV compared to women (Malawi: 30% vs. 17%, p < 0.0001; Zimbabwe: 35% vs. 19%, p < 0.0001; Lesotho: 34% vs. 15%, p < 0.0001; Zambia: 36% vs. 20%, p < 0.0001); although, less of a differential existed in Ethiopia (54% vs. 56%, p = 0.12) and Rwanda (19% vs. 14%, p < 0.0001). When offered a test during the survey, 85% to 99% of sexually active men who reported never previously testing, accepted testing. HIV positivity ranged from <0.05% to 14% for never tested men. After adjusting for age, factors associated with never having tested for HIV were never being married (aOR range: 1.46 to 10.39), not having children (aOR: 1.36 to 3.59) and lower education (less than primary education aOR: 2.77 to 5.59). CONCLUSIONS: Although higher proportions of men than women had never tested for HIV, 85% to 99% of men did accept a test when offered. Finding opportunities to offer HIV testing to single men without children, older men who have never tested, and those disadvantaged with less schooling and employment, alongside other facility and community-based services, will be important in identifying those living with undiagnosed HIV and improving men's health.

Retest and treat: a review of national HIV retesting guidelines to inform elimination of mother-to-child HIV transmission (EMTCT) efforts.

INTRODUCTION: High maternal HIV incidence contributes substantially to mother-to-child HIV transmission (MTCT) in some settings. Since 2006, HIV retesting during the third trimester and breastfeeding has been recommended by the World Health Organization in higher prevalence (≥5%) settings to reduce MTCT. However, many countries lack clarity on when and how often to retest pregnant and postpartum women to optimize resources and service delivery. We reviewed and characterized national guidelines on maternal retesting based on timing and frequency. METHODS: We identified 52 countries to represent variations in HIV prevalence, geography, and MTCT priority and searched available national MTCT, HIV testing and HIV treatment policies published between 2007 and 2017 for recommendations on retesting during pregnancy, labour/delivery and postpartum. Recommended retesting frequency and timing was extracted. Country HIV prevalence was classified as: very low (<1%), low (1% to 5%), intermediate (>5 to <15%) and high (≥15%). Women with unknown HIV status at delivery/postpartum were included in retesting guidelines. RESULTS AND DISCUSSION: Overall, policies from 49 countries were identified; 51% from 2015 or later and most (n = 25) were from Africa. Four countries were high HIV prevalence, seven intermediate, sixteen low and twenty-two very low. Most (n = 31) had guidance on universal voluntary opt-out HIV testing at the first antenatal care (ANC) visit. Beyond the first ANC visit, the majority (78%, n = 38) had guidance on retesting; 22 recommended retesting all women with unknown/negative status, five only if unknown HIV status, three in pregnancy based on risk and eight combining these approaches. Retesting was universally recommended during pregnancy, labour/delivery, and postpartum for all high prevalence settings and four of seven intermediate prevalence settings. Five UNAIDS priority countries for EMTCT with low/very low HIV prevalence, but high/intermediate MTCT, had no guidance on retesting. CONCLUSIONS: Retesting guidelines for pregnant and postpartum women were ubiquitous in high prevalence countries and defined in some intermediate prevalence countries, but absent in some low HIV prevalence countries with high MTCT. Countries may require additional guidance on how to optimize maternal HIV testing and whether to prioritize retesting efforts or discontinue universal retesting based on HIV incidence. Research is needed to assess country-level guideline implementation and impact.

Dynamic regulation of HIV-1 capsid interaction with the restriction factor TRIM5α identified by magic-angle spinning NMR and molecular dynamics simulations.

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

HIV Partner Notification Values and Preferences Among Sex Workers, Fishermen, and Mainland Community Members in Rakai, Uganda: A Qualitative Study.

HIV partner notification involves contacting sexual partners of people who test HIV positive and referring them to HIV testing, treatment, and prevention services. To understand values and preferences of key and general populations in Rakai, Uganda, we conducted 6 focus group discussions and 63 in-depth interviews in high prevalence fishing communities and low prevalence mainland communities. Participants included fishermen and sex workers in fishing communities, male and female mainland community members, and healthcare providers. Questions explored three approaches: passive referral, provider referral, and contract referral. Qualitative data were coded and analyzed using a team-based matrix approach. Participants agreed that passive referral was most suitable for primary partners. Provider referral was acceptable in fishing communities for notifying multiple, casual partners. Healthcare providers voiced concerns about limited time, resources, and training for provider-assisted approaches. Options for partner notification may help people overcome barriers to HIV serostatus disclosure and help reach key populations.

Quenching protein dynamics interferes with HIV capsid maturation.

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA-SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA-SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix-coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

MAS NMR of HIV-1 protein assemblies.

The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.