Faculty Bibliography

Membrane proteins are involved in all cellular processes from signaling cascades to nutrient uptake and waste disposal. Because of these essential functions, many membrane proteins are recognized as important, yet elusive, clinical targets. Recent advances in structural biology have answered many questions about how membrane proteins function, yet one of the major bottlenecks remains the ability to obtain sufficient quantities of pure and homogeneous protein. This is particularly true for human membrane proteins, where novel expression strategies and structural techniques are needed to better characterize their function and therapeutic potential. One way to approach this challenge is to determine the structure of smaller pieces of membrane proteins that can be assembled into models of the complete protein. This unit describes the rationale for working with single or multiple transmembrane segments and provides a description of strategies and methods to express and purify them for structural and functional studies using a maltose binding protein (MBP) fusion. The bulk of the unit outlines a detailed methodology and justification for producing these peptides under native-like conditions.

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-A decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.

Salt stress is a widespread phenomenon, limiting plant performance in large areas around the world. Although various types of plant sodium/proton antiporters have been characterized, the physiological function of NHD1 from Arabidopsis thaliana has not been elucidated in detail so far. Here we report that the NHD1-GFP fusion protein localizes to the chloroplast envelope. Heterologous expression of AtNHD1 was sufficient to complement a salt-sensitive Escherichia coli mutant lacking its endogenous sodium/proton exchangers. Transport competence of NHD1 was confirmed using recombinant, highly purified carrier protein reconstituted into proteoliposomes, proving Na(+) /H(+) antiport. In planta NHD1 expression was found to be highest in mature and senescent leaves but was not induced by sodium chloride application. When compared to wild-type controls, nhd1 T-DNA insertion mutants showed decreased biomasses and lower chlorophyll levels after sodium feeding. Interestingly, if grown on sand and supplemented with high sodium chloride, nhd1 mutants exhibited leaf tissue Na(+) levels similar to those of wild-type plants, but the Na(+) content of chloroplasts increased significantly. These high sodium levels in mutant chloroplasts resulted in markedly impaired photosynthetic performance as revealed by a lower quantum yield of photosystem II and increased non-photochemical quenching. Moreover, high Na(+) levels might hamper activity of the plastidic bile acid/sodium symporter family protein 2 (BASS2). The resulting pyruvate deficiency might cause the observed decreased phenylalanine levels in the nhd1 mutants due to lack of precursors.

Voluntary medical male circumcision (VMMC) is capable of reducing the risk of sexual transmission of HIV from females to males by approximately 60%. In 2007, the WHO and the Joint United Nations Programme on HIV/AIDS (UNAIDS) recommended making VMMC part of a comprehensive HIV prevention package in countries with a generalized HIV epidemic and low rates of male circumcision. Modeling studies undertaken in 2009-2011 estimated that circumcising 80% of adult males in 14 priority countries in Eastern and Southern Africa within five years, and sustaining coverage levels thereafter, could avert 3.4 million HIV infections within 15 years and save US$16.5 billion in treatment costs. In response, WHO/UNAIDS launched the Joint Strategic Action Framework for accelerating the scale-up of VMMC for HIV prevention in Southern and Eastern Africa, calling for 80% coverage of adult male circumcision by 2016. While VMMC programs have grown dramatically since inception, they appear unlikely to reach this goal. This review provides an overview of findings from the PLOS Collection "Voluntary Medical Male Circumcision for HIV Prevention: Improving Quality, Efficiency, Cost Effectiveness, and Demand for Services during an Accelerated Scale-up." The use of devices for VMMC is also explored. We propose emphasizing management solutions to help VMMC programs in the priority countries achieve the desired impact of averting the greatest possible number of HIV infections. Our recommendations include advocating for prioritization and funding of VMMC, increasing strategic targeting to achieve the goal of reducing HIV incidence, focusing on programmatic efficiency, exploring the role of new technologies, rethinking demand creation, strengthening data use for decision-making, improving governments' program management capacity, strategizing for sustainability, and maintaining a flexible scale-up strategy informed by a strong monitoring, learning, and evaluation platform.

Scaling up HIV prevention programming among key populations (female sex workers and men who have sex with men) has been a central strategy of the Government of India. However, state governments have lacked the technical and managerial capacity to oversee and scale up interventions or to absorb donor-funded programs. In response, the national government contracted Technical Support Units (TSUs), teams with expertise from the private and nongovernmental sectors, to collaborate with and assist state governments. In 2008, a TSU was established in Karnataka, one of 6 Indian states with the highest HIV prevalence in the country and where monitoring showed that its prevention programs were reaching only 5% of key populations. The TSU provided support to the state in 5 key areas: assisting in strategic planning, rolling out a comprehensive monitoring and evaluation system, providing supportive supervision to intervention units, facilitating training, and assisting with information, education, and communication activities. This collaborative management model helped to increase capacity of the state, enabling it to take over funding and oversight of HIV prevention programs previously funded through donors. With the combined efforts of the TSU and the state government, the number of intervention units statewide increased from 40 to 126 between 2009 and 2013. Monthly contacts with female sex workers increased from 5% in 2008 to 88% in 2012, and with men who have sex with men, from 36% in 2009 to 81% in 2012. There were also increases in the proportion of both populations who visited HIV testing and counseling centers (from 3% to 47% among female sex workers and from 6% to 33% among men who have sex with men) and sexually transmitted infection clinics (from 4% to 75% among female sex workers and from 7% to 67% among men who have sex with men). Changes in sexual behaviors among key populations were also documented. For example, between 2008 and 2010, the proportion of surveyed female sex workers in 9 districts reporting that they used a condom at last intercourse rose from 60% to 68%; in 6 districts, the proportion of surveyed men who have sex with men reporting that they used a condom at last anal sex increased from 89% to 97%. The Karnataka experience suggests that TSUs can help governments enhance managerial and technical resources and leverage funds more effectively. With careful management of the working and reporting relationships between the TSU and the state government, this additional capacity can pave the way for the government to improve and scale up programs and to absorb previously donor-funded programs.

BACKGROUND AND METHODS: By December 2013, it was estimated that close to 6 million men had been circumcised in the 14 priority countries for scaling up voluntary medical male circumcision (VMMC), the majority being adolescents (10-19 years). This article discusses why efforts to scale up VMMC should prioritize adolescent men, drawing from new evidence and experiences at the international, country, and service delivery levels. Furthermore, we review the extent to which VMMC programs have reached adolescents, addressed their specific needs, and can be linked to their sexual and reproductive health and other key services. RESULTS AND DISCUSSION: In priority countries, adolescents represent 34%-55% of the target population to be circumcised, whereas program data from these countries show that adolescents represent between 35% and 74% of the circumcised men. VMMC for adolescents has several advantages: uptake of services among adolescents is culturally and socially more acceptable than for adults; there are fewer barriers regarding sexual abstinence during healing or female partner pressures; VMMC performed before the age of sexual debut has maximum long-term impact on reducing HIV risk at the individual level and consequently reduces the risk of transmission in the population. Offered as a comprehensive package, adolescent VMMC can potentially increase public health benefits and offers opportunities for addressing gender norms. Additional research is needed to assess whether current VMMC services address the specific needs of adolescent clients, to test adapted tools, and to assess linkages between VMMC and other adolescent-focused HIV, health, and social services.

Understanding the neural mechanisms underlying learning and memory in the entorhinal-hippocampal circuit is a central challenge of systems neuroscience. For more than 40 years, electrophysiological recordings in awake, behaving animals have been used to relate the receptive fields of neurons in this circuit to learning and memory. However, the vast majority of such studies are purely observational, as electrical, surgical, and pharmacological circuit manipulations are both challenging and relatively coarse, being unable to distinguish between specific classes of neurons. Recent advances in molecular genetic tools can overcome many of these limitations, enabling unprecedented control over neural activity in behaving animals. Expression of pharmaco- or optogenetic transgenes in cell-type-specific "driver" lines provides unparalleled anatomical and cell-type specificity, especially when delivered by viral complementation. Pharmacogenetic transgenes are specially designed neurotransmitter receptors exclusively activated by otherwise inactive synthetic ligands and have kinetics similar to traditional pharmacology. Optogenetic transgenes use light to control the membrane potential, and thereby operate at the millisecond timescale. Thus, activation of pharmacogenetic transgenes in specific neuronal cell types while recording from other parts of the circuit allows investigation of the role of those neurons in the steady state, whereas optogenetic transgenes allow one to determine the immediate network response.