Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances.

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

Structural insight into proteorhodopsin oligomers.

Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor.

Herein, we identify the coordination environment of Cu²(+) in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²(+) and Cu²(+) significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²(+) can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²(+) is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²(+) coordination and stoichiometry. We show that Cu²(+) has one binding site per α1 subunit, and that five Cu²(+) can be coordinated per GlyR. Cu²(+) binds to E192 and H215 in each subunit of GlyR with a 40 µM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²(+) coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²(+) site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

Consistent condom use is associated with lower prevalence of human papillomavirus infection in men.

INTRODUCTION: Reported associations between condom use and human papillomavirus (HPV) infection in men have been inconsistent. METHODS: We tested 463 men, ages 18-40 years, in 2 cities in the United States for 37 HPV types in samples from 5 anogenital sites. Men answered questionnaires regarding number of partners and frequency of condom use during vaginal sex in the past 3 months (5 categories, from "always" to "never"). Among 393 men who reported > or =1 female partner in the past 3 months, the proportions of men with HPV detected overall and at each anatomic site by frequency of condom use were calculated. Logistic regression was used to examine associations between frequency of condom use and HPV detection. Effect modification by number of recent partners (1 vs >1) was evaluated. RESULTS: The proportion of men positive for HPV ranged from 37.9% among men who reported they "always" used condoms to 53.9% among those who reported they "never" used condoms (P for trend = .008). Always using condoms (vs using them less frequently) was associated with lower odds of HPV detection (adjusted odds ratio, 0.50 [95% confidence interval, 0.30-0.83]). This association was stronger among men with >1 partner than among men with only 1 partner (P for interaction = .05). CONCLUSIONS: Consistent condom use was strongly associated with lower HPV prevalence in men.

Risk factors for incident herpes simplex type 2 virus infection among women attending a sexually transmitted disease clinic.

OBJECTIVES: To estimate the incidence of herpes simplex type 2 virus (HSV-2) infection, to identify risk factors for its acquisition, and to assess the protective effect of condoms. STUDY DESIGN: Prospective study of 293 HSV-2 seronegative women, aged 18 to 35 years, attending a sexually transmitted disease clinic in Alabama from 1992 to 1995. RESULTS: Incidence of HSV-2 infection was 20.5 per 100 woman-years [95% confidence interval (CI), 13.1-30.5]. Young women (18-20 years) had a significantly higher risk of incident HSV-2 infection [adjusted hazard ratio (HR), 2.8; 95% CI, 1.3-6.4] than older women. Women diagnosed with prevalent or incident bacterial vaginosis had a higher incidence of HSV-2 infection than those who were not so diagnosed (adjusted HR, 2.4; 95% CI, 1.1-5.6). No significant protective effect was observed for consistent (100%) condom use without breakage and slippage against HSV-2 acquisition (adjusted HR, 0.8; 95% CI, 0.2-2.3). CONCLUSION: Acquisition of HSV-2 infection among study participants was higher than previous estimates for adult female sexually transmitted disease clinic attendees, and no protective effect for condoms was demonstrated. The high incidence of HSV-2 infection with its potential for adverse health consequences emphasizes the need for better prevention strategies.

Testing for sexually transmitted diseases in U.S. Public health laboratories in 2004.

OBJECTIVE: Appropriate laboratory testing practices are a critical part of sexually transmitted disease (STD) control. GOAL: The goal of this study was to describe the type and volume of STD tests performed in public health laboratories in the United States in 2004. STUDY DESIGN: A web-based survey was made available to 144 members of the Association of Public Health Laboratories. RESULTS: One hundred fourteen laboratories responded (79%). Overall, 3,553,196 chlamydia tests and 3,461,151 gonorrhea tests were performed; 64.4% of chlamydia tests and 60.8% of gonorrhea tests were nucleic acid amplification tests. Ninety-four percent of laboratories performed syphilis testing. Few laboratories used type-specific tests for herpes simplex virus or used new tests for trichomoniasis, bacterial vaginosis, or human papillomavirus. CONCLUSIONS: This survey collected important data that can be used to monitor trends in STD testing practices in public health laboratories.

Prevalence of HPV infection among men: A systematic review of the literature.

BACKGROUND: Human papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection; an estimated 6.2 million persons are newly infected every year in the United States. There are limited data on HPV infection in heterosexual men. METHODS: We conducted a systematic review of the literature by searching MEDLINE using the terms "human papillomavirus," "HPV," "male," "seroprevalence," and "serology" to retrieve articles published from 1 January 1990 to 1 February 2006. We included studies that had data on population characteristics and that evaluated male genital anatomic sites or specimens for HPV DNA or included assessments of seropositivity to HPV type 6, 11, 16, or 18 in men. We excluded studies that had been conducted only in children or immunocompromised persons (HIV infected, transplant recipients, or elderly). RESULTS: We included a total of 40 publications on HPV DNA detection and risk factors for HPV in men; 27 evaluated multiple anatomic sites or specimens, 10 evaluated a single site or specimen, and 3 evaluated risk factors or optimal anatomic sites/specimens for HPV detection. Twelve studies assessed site- or specimen-specific HPV DNA detection. HPV prevalence in men was 1.3%-72.9% in studies in which multiple anatomic sites or specimens were evaluated; 15 (56%) of these studies reported > or =20% HPV prevalence. HPV prevalence varied on the basis of sampling, processing methods, and the anatomic site(s) or specimen(s) sampled. We included 15 publications reporting HPV seroprevalence. Rates of seropositivity depended on the population, HPV type, and methods used. In 9 studies that evaluated both men and women, all but 1 demonstrated that HPV seroprevalence was lower in men than in women. CONCLUSION: HPV infection is highly prevalent in sexually active men and can be detected by use of a variety of specimens and methods. There have been few natural-history studies and no transmission studies of HPV in men. The information that we have reviewed may be useful for future natural-history studies and for modeling the potential impact of a prophylactic HPV vaccine.

Condom use and risk of gonorrhea and Chlamydia: a systematic review of design and measurement factors assessed in epidemiologic studies.

BACKGROUND: Studies of condom use to reduce risk of most sexually transmitted infection provide inconsistent results. This inconsistency is often attributed to methodologic limitations yet has not been assessed systematically. OBJECTIVES: The objectives of this study were to review studies of condom use and risk of gonorrhea and chlamydia, and to evaluate the importance of 4 key design and measurement factors on condom effectiveness estimates. DESIGN: We reviewed studies published 1966-2004 to assess risk reduction for gonorrhea and/or chlamydia associated with male condom use. RESULTS: Of 45 studies identified, most found reduced risk of infection associated with condom use. All studies reviewed had methodologic limitations: only 28 (62%) distinguished consistent from inconsistent use; 2 (4%) reported on correct use or use problems; 13 (29%) distinguished incident from prevalent infection; and one (2%) included a population with documented exposure to infection. Eight of 10 studies with 2 or more of these attributes reported statistically significant protective effects for condom use versus 15 of 35 studies with zero or one attribute (80% vs. 43%, P = 0.04). CONCLUSIONS: Condom use was associated with reduced risk of gonorrhea and chlamydia in men and women in most studies, despite methodologic limitations that likely underestimate condom effectiveness. Epidemiologic studies that better address these factors are needed to provide more accurate assessment of condom effectiveness.

Seroprevalence of human papillomavirus type 16 in children.

We evaluated the prevalence of antibodies to human papillomavirus (HPV) type 16 in a representative sample of children 6-11 years of age in the United States. Serum samples and questionnaire data were collected between 1991 and 1994, for the National Health and Nutrition Examination Survey III. HPV-16-specific immunoglobulin G antibodies were detected by an HPV-16 L1 virus-like particle-based enzyme-linked immunosorbant assay. Overall, 2.4% of 1316 children 6-11 years of age were seropositive. Seroprevalence was higher in boys than in girls (3.5% vs. 1.2%; P=.08) and in children >7 years of age than in children < or =7 years of age (3.3% vs. 0.4%; P<.05). None of the variables tested for, including race/ethnicity, socioeconomic status, and urban or rural residence, were significantly associated with HPV-16 seropositivity. To explain HPV-16 seropositivity in this population, further study is required.

Return for results after herpes simplex virus type 2 screening.

OBJECTIVE: The objective of this study was to evaluate factors associated with return for results of type-specific herpes simplex virus (HSV) screening. METHODS: Participants receiving type-specific HSV testing were asked to return for results 2 weeks after testing. Predictors of return included demographics, herpes-related knowledge and attitudes, and past sexual behaviors. RESULTS: A total of 820 sexually active subjects (age, 14-30 years; 41% male) received HSV screening and 578 (70%) returned for results. Higher probability of return for HSV testing results was significantly associated with older age (odds ratio [OR], 1.06), female gender (OR, 1.57), enrollment at sites other than the county sexually transmitted disease clinic (OR, 1.70-4.71), and heightened level of perceived HSV vulnerability (OR, 1.07). Lower probability of return was associated with having more than 1 recent sex partner (OR, 0.46). CONCLUSIONS: Lower rates of return of high-risk patients suggest the need to focus resources on receipt of test results.

Factors predicting the acceptance of herpes simplex virus type 2 antibody testing among adolescents and young adults.

BACKGROUND: The rates and determinants of acceptance of herpes simplex virus type 2 (HSV-2) testing have not been adequately studied. OBJECTIVES: The objective of this study was to identify factors associated with acceptance of HSV-2 antibody testing in individuals with no history of genital herpes. STUDY: We conducted a cross-sectional survey study followed by the offer of free HSV-2 serologic testing at an urban sexually transmitted disease (STD) clinic, 2 general adult medical clinics, an urban university campus, and an urban adolescent medicine clinic. A total of 1199 individuals aged 14 to 30 years completed the survey and were offered testing. RESULTS: A total of 68.4% accepted HSV-2 testing. Factors independently associated with acceptance were female sex, older age, having an STD history, having 1 or more sexual partners in the last 6 months, perceived vulnerability to HSV-2 infection, and perceived benefits of HSV-2 testing. Fear of needles predicted rejection of testing, as did attending a general medical clinic versus an STD clinic and nonwhite race. CONCLUSION: There is a substantial interest in HSV-2 antibody testing across a variety of settings. Those at greatest behavioral and historic risk for HSV-2 infection, women, and persons whose health beliefs are consistent with testing are more likely to accept serologic testing when it is offered.

Predictors of herpes simplex virus type 2 antibody positivity among persons with no history of genital herpes.

BACKGROUND: The demographic, historical, and behavioral factors that predict a positive herpes simplex virus type 2 (HSV-2) antibody test in persons without a history of genital herpes have not been well-defined. METHODS: Individuals (age 14-30 years) without a history of genital herpes completed a questionnaire and were offered free HSV-2 antibody testing. Factors from the questionnaire were correlated with the HSV-2 antibody result. RESULTS: Univariate analysis showed that female gender was significantly associated with positive test results. In gender-specific, multiple logistic regression models, a positive HSV-2 antibody test among men was associated with older age, non-white race, and a history of sexually transmitted disease (STD). Gender-specific symptom scores from the questionnaire were not predictive in either gender, but the gender-common symptom score was marginally predictive of a positive HSV-2 antibody test in women. Among women, older age, non-white race, and STD history predicted a positive test. CONCLUSIONS: Among young persons with no history of genital herpes who agreed to HSV-2 antibody testing, increasing age, non-white race, and a history of an STD were predictors of a positive test. A history of frequent pain, itching, burning, and rashes in the anogenital region was marginally associated with positive HSV-2 tests in women. These results might help guide selective use of HSV-2 antibody screening.

Pregnancy outcomes following systemic prenatal acyclovir exposure: Conclusions from the international acyclovir pregnancy registry, 1984-1999.

BACKGROUND: Oral acyclovir is commonly used for genital herpes and other herpesvirus infections. Data on potential fetal risk are extremely limited. From 1984 to 1998, the Acyclovir in Pregnancy Registry monitored birth outcomes of women exposed to oral or intravenous acyclovir during pregnancy. This report describes the final results. METHODS: The registry was publicized to health care providers most likely to diagnose pregnancy; providers called the registry telephone number, then mailed in a brief questionnaire. Pregnancy outcomes were categorized either as outcomes with birth defects or outcomes without birth defects, subcategorized as live births, spontaneous pregnancy losses (including stillbirths), and induced abortions. Birth defects were defined using a modification of the CDC definition for birth defects surveillance systems. Observed rates were compared to the rate (3.2%) of birth defects expected in the general population. RESULTS: Between June 1, 1984 and June 30, 1998, 1695 pregnancies exposed to oral or IV acyclovir were registered; 461 (27%) were lost to follow-up. A total of 1234 pregnancies in 24 countries were followed, with a total of 1246 outcomes. Among 1246 pregnancy outcomes, 756 involved acyclovir exposure in the first trimester, 197 in the second trimester, and 291 in the third trimester. Among live births with first trimester acyclovir exposure, risk of birth defects was 19 of 596 (3.2%; 95% CI, 2.0-5.0%). No unusual defects or pattern of defects were apparent. CONCLUSIONS: The observed rates and types of birth defects for pregnancies exposed to acyclovir did not differ significantly from those in the general population. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.

Laboratory tests used in US public health laboratories for sexually transmitted diseases, 2000.

BACKGROUND AND OBJECTIVES: Public health laboratories are a critical component of sexually transmitted disease (STD) control in the United States. GOAL: The goal of this study was to describe the types and volume of STD tests performed in U.S. public health laboratories in 2000. STUDY DESIGN: A survey was mailed to 123 members of the Association of Public Health Laboratories. RESULTS: Eighty-one percent of 100 laboratories responded. Overall, 3294739 chlamydia tests and 3088142 gonorrhea tests were done; 62.4% of chlamydia tests and 63.6% of gonorrhea tests were DNA probes. Fifty-six percent of laboratories performed rapid plasma reagin (RPR) tests and 55% performed Venereal Disease Research Laboratory (VDRL) tests; the number of RPR tests performed was twice that of VDRL tests. Few laboratories used new technologies for bacterial vaginosis and trichomoniasis. Eighteen percent of laboratories performed herpes simplex virus serology; however, most used inaccurate tests. No laboratories performed human papillomavirus tests. CONCLUSIONS: This survey documents for the first time STD tests performed in U.S. public health laboratories.

Seroprevalence of human papillomavirus type 16 infection in the United States.

Infection with human papillomavirus (HPV) type 16 accounts for about half of cervical cancers worldwide. This study investigated the seroepidemiology of HPV-16 infection in the United States by using a population-based survey. Serum samples and questionnaire data were collected from 1991 to 1994 for the National Health and Nutrition Examination Surveys. HPV-16-specific IgG antibody was detected by use of an HPV-16 virus-like particle ELISA. HPV-16 seropositivity in the US population aged 12-59 years was 13.0% (95% confidence interval, 11.5%-14.7%). Seroprevalence was higher in women (17.9%) than in men (7.9%). Age, race/ethnicity, and number of lifetime sex partners were associated with HPV seropositivity in women. Race/ethnicity, age at first intercourse, urban/nonurban residence, years of sexual activity, and having had sex with a man were associated with HPV seropositivity in men. Information on HPV-16 seroepidemiology will be important for designing prevention efforts including vaccine programs.