The Puerto Rico Young Adults' Stress, Contextual, Behavioral & Cardiometabolic Risk (PR-OUTLOOK) Study: Design and Methods.

The Puerto Rico (PR) Young Adults' Stress, Contextual, Behavioral & Cardiometabolic Risk Study (PR-OUTLOOK) is investigating overall and component-specific cardiovascular health (CVH) and cardiovascular disease (CVD) risk factors in a sample of young (age 18-29) Puerto Rican adults in PR (target n=3,000) and examining relationships between individual-, family/social- and neighborhood-level stress and resilience factors and CVH and CVD risk factors. The study is conducting standardized measurements of CVH and CVD risk factors and demographic, behavioral, psychosocial, neighborhood, and contextual variables and establishing a biorepository of blood, saliva, urine, stool, and hair samples. The assessment methods are aligned with other National Heart, Lung, and Blood Institute funded studies: the Puerto Rico Observational Study of Psychosocial, Environmental, and Chronic Disease Trends (PROSPECT) of adults 30-75 years, the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), the Boston Puerto Rican Health Study (BPRHS), and the Coronary Artery Risk Development in Young Adults (CARDIA). PR-OUTLOOK data and its biorepository will facilitate future longitudinal studies of the temporality of associations between stress and resilient factors and CVH and CVD risk factors among young Puerto Ricans, with remarkable potential for advancing the scientific understanding of these conditions in a high-risk but understudied young population.

Gendered Race and Setting Matter: Sources of Complexity in the Relationships Between Reported Interpersonal Discrimination and Cardiovascular Health in the CARDIA Study.

OBJECTIVES: Using data from black and white adults enrolled in a community-based, multi-city cohort assembled in the mid-1980s, we examined whether reported experiences of interpersonal racial and gender discrimination differentially impacted on future cardiovascular health (CVH) depending on gendered race and the setting in which the interactions were reported to have occurred. METHODS: Discrimination in eight possible settings was assessed using the Experiences of Discrimination scale at year 7; CVH two decades later was examined using a modified Life's Simple 7 score, with higher scores indicating better health. Separate multivariable linear regressions evaluated the associations between reports of racial and gender discrimination and CVH score in each possible setting stratified by gendered race. RESULTS: Mean (SD) CVH scores at year 30 were 7.8(1.9), 8.1(1.8), 8.9(2. 0), and 8.8(1.8) among black women, black men, white women, and white men, respectively. For black women, reporting both racial and gender discrimination while receiving medical care was associated with lower CVH score. Among black men, reporting both forms of discrimination while getting a job, at work, at school, and receiving medical care was associated with lower CVH score. Among whites, reported discrimination while obtaining housing and by the police or courts (women), and in public and at work (men), was associated with a lower CVH score. CONCLUSIONS: The setting in which discrimination is reported may be an important indicator of whether discriminatory experiences are negatively associated with CVH, providing insight on distinct effect pathways among black and white women and men.

Intersectional effects of racial and gender discrimination on cardiovascular health vary among black and white women and men in the CARDIA study.

Testing hypotheses from the emerging Identity Pathology (IP) framework, we assessed race-gender differences in the effects of reporting experiences of racial and gender discrimination simultaneously compared with racial or gender discrimination alone, or no discrimination, on future cardiovascular health (CVH). Data were from a sample of 3758 black or white adults in CARDIA, a community-based cohort recruited in Birmingham, AL; Chicago, IL; Minneapolis, MN, and Oakland, CA in 1985-6 (year 0). Racial and gender discrimination were assessed using the Experiences of Discrimination scale. CVH was evaluated using a 12-point composite outcome modified from the Life's Simple 7, with higher scores indicating better health. Multivariable linear regressions were used to evaluate the associations between different perceptions of discrimination and CVH scores two decades later by race and gender simultaneously. Reporting racial and gender discrimination in ≥2 settings were 48% of black women, 42% of black men, 10% of white women, and 5% of white men. Year 30 CVH scores (mean, SD) were 7.9(1.4), 8.1(1.6), 8.8(1.6), and 8.7(1.3), respectively. Compared with those of their race-gender groups reporting no discrimination, white women reporting only gender-based discrimination saw an adjusted score difference of +0.3 (95% CI: 0.0,0.6), whereas white men reporting only racial discrimination had on average a 0.4 (95% CI: 0.1,0.8) higher score, and scores among white men reporting both racial and gender discrimination were on average 0.6 (95% CI: 1.1,-0.1) lower than those of their group reporting no discrimination. Consistent with predictions of the IP model, the associations of reported racial and gender discrimination with future CVH were different for different racially-defined gender groups. More research is needed to understand why reported racial and gender discrimination might better predict deterioration in CVH for whites than blacks, and what additional factors associated with gender and race contribute variability to CVH among these groups.

Improvement of heat transfer by means of ultrasound: Application to a double-tube heat exchanger.

A new kind of ultrasonically-assisted heat exchanger has been designed, built and studied. It can be seen as a vibrating heat exchanger. A comprehensive description of the overall experimental set-up is provided, i.e. of the test rig and the acquisition system. Data acquisition and processing are explained step-by-step with a detailed example of graph obtained and how, from these experimental data, energy balance is calculated on the heat exchanger. It is demonstrated that ultrasound can be used efficiently as a heat transfer enhancement technique, even in such complex systems as heat exchangers.

Socio-demographic determinants of aflatoxin B1-lysine adduct levels among pregnant women in Kumasi, Ghana.

OBJECTIVES: Aflatoxins are fungal metabolites that contaminate staple food crops in many developing countries. Although studies have linked these toxins to adverse birth outcomes and poor infant development, no study has investigated the socio-demographic and economic determinants of aflatoxin levels among pregnant women living in sub-Saharan Africa. DESIGN: A cross-sectional study was conducted among 785 pregnant women in Kumasi. Aflatoxin B1 lysine adduct levels (AF-ALB) were determined by High Performance Liquid Chromatography. Analysis of variance was used to determine mean log AF-ALB levels and significance of differences in these levels according to socio-demographic variables. Logistic regression was used to identify independent associations of socio-demographics with having AF-ALB levels (≥ 11.34 pg/mg; upper quartile). RESULTS: AF-ALB levels ranged from 0.44 pg/mg to 268.73 pg/mg albumin with a median level of 5.0 pg/mg. Bivariate analyses indicates that mean ln AF-ALB as well as the percent of women having high AF-ALB levels (≥ 11.34 pg/mg; upper quartile) were inversely associated with indices of higher socioeconomic status: higher education and income, being employed and having a flush toilet. Higher income, being employed, having one child (verses no children) and having a flush toilet (verses no toilet facilities) were each independently associated with a 30-40% reduced odds of high AF-ALB levels. CONCLUSIONS: Additional research is needed to investigate how socio-demographic and economic factors interact to influence aflatoxin ingestion by individuals in regions with high aflatoxin crop contamination. This knowledge can be used to formulate and implement policies that will reduce exposure of women and their unborn children to these toxins.

Demographic and geographic variations of oral health among African Americans based on NHANES III.

UNLABELLED: As efforts continue to improve the health of all US citizens, oral health must not be overlooked. Oral health is an integral part of overall health status and oral diseases are among the most prevalent of all health problems. OBJECTIVES: To describe the oral health status and oral health behaviors of African Americans. METHODS: The National Health and Nutrition Examination Survey (NHANES III) data set was used to examine a range of oral health indicators of African Americans with specific attention to demographic and geographic factors. The original data set consisted of 20,050 subjects, gathered through the use of complex, multi-stage, stratified and clustered sampling techniques. Only African Americans were included in this study which resulted in a sample of 5,616. Statistical analysis was conducted to allow the proper modeling of the complex, stratified, multistage survey design and sample weights of NHANES III. RESULTS: Sixty-two percent of respondents indicated that they only visit the dentist when needed and had no regular visitation schedule. Dental health was worse for those individuals who were poor, unemployed, and uninsured. Regional differences in dental care appeared with individuals living in the south reporting poorer dental health. CONCLUSIONS: The findings from this study are useful for identifying sociodemographic and geographic factors related to oral health status. The insights gained from this study illustrate the need for tailoring oral health promotion programmes and services to specific groups within the African American community because service utilisation and response patterns and perceptions may be different.

A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation.

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.

Validity of self-reported sexually transmitted diseases among African American female adolescents participating in an HIV/STD prevention intervention trial.

BACKGROUND: Studies assessing the validity attributed to self-reported measures of sexually transmitted diseases (STDs) clearly are needed, particularly those used for high-risk populations such as female adolescents, in whom STD prevention is a priority. GOAL: To determine the accuracy of self-reported STD test results in female adolescents over a relatively brief period ( approximately 28 days). STUDY DESIGN: A prospective, randomized, controlled clinical trial of STD/HIV prevention for African American females, ages 14 to 18, was conducted. Study participants were recruited from medical clinics and school health classes in low-income neighborhoods of Birmingham, Alabama, that had high rates of unemployment, substance abuse, violence, STDs, and teenage pregnancy. RESULTS: Of the 522 adolescents enrolled in the trial, 92% (n = 479) completed baseline STD testing and follow-up surveys. At baseline, 28% had positive test results for at least one disease: 4.8% for Neisseria gonorrhoeae, 17.1% for Chlamydia trachomatis, and 12.3% for Trichomonas vaginalis. Of the adolescents with negative STD test results, 98.8% were accurate in their self-report of STD status, as compared with 68.7% of the adolescents with positive results. Underreporting varied by type of STD. Adolescents who accurately reported their positive STD status were significantly more likely to report their receipt of treatment accurately (P < 0.001). CONCLUSIONS: The substantial underreporting of STD incidence in this study suggests that reliance on self-reports of STD history may introduce misclassification bias, potentially leading to false conclusions regarding the efficacy of prevention interventions. This observation highlights the importance of using biologic indicators as outcome measures.

Mutations in the N-terminus of VP5 alter its interaction with the scaffold proteins of herpes simplex virus type 1.

During the assembly process of herpes simplex virus type 1 capsids, there is an essential interaction between the C-terminal tail of the scaffold proteins (22a and 21) and the major capsid protein (VP5). Recent studies of spontaneous revertant viruses that overcome a blocked maturation cleavage site of the scaffold proteins have shown that the N-terminus of VP5 is important for this interaction. One of the revertant viruses, PR7, encodes a second-site mutation at residue 69 of VP5 which unlike wild-type VP5 fails to interact with 22a and thus gives white colonies in the yeast two-hybrid assay. In the present study a small DNA fragment, encoding residues 1 to 85 of wild-type and PR7 VP5, was mutagenized using error-prone PCR. Mutagenized DNA was used in the yeast two-hybrid assay to identify mutations in wild-type VP5 that resulted in loss of 22a binding (white colonies), or in PR7 VP5 that resulted in a gain of function (blue colonies). For the loss of function experiments, using KOS VP5, a row of eight thymidine nucleotides (codons 37-40) resulted in many frameshift mutations, which led us to terminate the study without reaching a statistically significant result. For the PR7 experiment, 30 clones were identified that had single amino acid substitutions, and these mutations were localized to amino acids 27-45 and 63-84 of VP5. The most frequent mutation was a reversion back to wild-type. The next most frequent were E28K and N63S, and these gave the highest beta-galactosidase enzyme activities (indicative of PR7VP5-22a interaction), 30 and 20% of wild-type, respectively. When E28K and N63S were transferred into the wild-type VP5 background, that is, in the absence of the PR7 mutation, they gave rise to different phenotypes. The E28K mutation lost its ability to interact with the scaffold proteins as judged by this assay. Therefore, it may be acting as a compensatory mutation whose phenotype is only expressed in the presence of the original PR7 mutation. However, the N63S mutation in the wild-type VP5 background increased the interaction, as judged by the beta-galactosidase activity, by a factor of 9 relative to when the PR7 mutation was present. Even more surprising, in the absence of the PR7 mutation the enzyme activity was still greater, by a factor of 2, than that observed for wild-type VP5. This study provides further evidence that the N-terminus of VP5 is in intimate association with the C-terminus of the scaffold proteins.

Improving quality improvement using achievable benchmarks for physician feedback: a randomized controlled trial.

CONTEXT: Performance feedback and benchmarking, common tools for health care improvement, are rarely studied in randomized trials. Achievable Benchmarks of Care (ABCs) are standards of excellence attained by top performers in a peer group and are easily and reproducibly calculated from existing performance data. OBJECTIVE: To evaluate the effectiveness of using achievable benchmarks to enhance typical physician performance feedback and improve care. DESIGN: Group-randomized controlled trial conducted in December 1996, with follow-up through 1998. SETTING AND PARTICIPANTS: Seventy community physicians and 2978 fee-for-service Medicare patients with diabetes mellitus who were part of the Ambulatory Care Quality Improvement Project in Alabama. INTERVENTION: Physicians were randomly assigned to receive a multimodal improvement intervention, including chart review and physician-specific feedback (comparison group; n = 35) or an identical intervention plus achievable benchmark feedback (experimental group; n = 35). MAIN OUTCOME MEASURE: Preintervention (1994-1995) to postintervention (1997-1998) changes in the proportion of patients receiving influenza vaccination; foot examination; and each of 3 blood tests measuring glucose control, cholesterol level, and triglyceride level, compared between the 2 groups. RESULTS: The proportion of patients who received influenza vaccine improved from 40% to 58% in the experimental group (P<.001) vs from 40% to 46% in the comparison group (P =.02). Odds ratios (ORs) for patients of achievable benchmark physicians vs comparison physicians who received appropriate care after the intervention, adjusted for preintervention care and nesting of patients within physicians, were 1.57 (95% confidence interval [CI], 1.26-1.96) for influenza vaccination, 1.33 (95% CI, 1.05-1.69) for foot examination, and 1.33 (95% CI, 1.04-1.69) for long-term glucose control measurement. For serum cholesterol and triglycerides, the achievable benchmark effect was statistically significant only after additional adjustment for physician characteristics (OR, 1.40 [95% CI, 1.08-1.82] and OR, 1.40 [95% CI, 1.09-1.79], respectively). CONCLUSION: Use of achievable benchmarks significantly enhances the effectiveness of physician performance feedback in the setting of a multimodal quality improvement intervention.

Second-site mutations encoding residues 34 and 78 of the major capsid protein (VP5) of herpes simplex virus type 1 are important for overcoming a blocked maturation cleavage site of the capsid scaffold proteins.

During assembly of the herpes simplex type 1 capsid, the major capsid protein VP5 interacts with the C-terminal residues of the scaffold proteins encoded by UL26 and UL26.5. Subsequent to capsid assembly the scaffold proteins are cleaved at the maturation site by a serine protease also encoded by UL26, thereby enabling the bulk of the scaffold proteins to be released from the capsid. Previously, a mutant virus (KUL26-610/611) was isolated in which this maturation cleavage site was blocked by replacing the Ala/Ser at the 610/611 cleavage site by Glu/Phe. This mutation was lethal and required a transformed cell line expressing wild-type UL26 gene products for growth. Although the mutation was lethal, spontaneous reversions occurred at a high frequency. Previously, a small number of revertants were isolated and all were found to have second-site mutations in VP5. The purpose of the present study was to do a comprehensive determination of the sites altered in VP5 by the second-site mutations. To do this, an additional 25 independent spontaneous revertants were characterized. Seven of the 25 arose by GC --> GT changes in codon 78, giving rise to an alanine to valine substitution. Four were the result of base changes at codon 34 but two different amino acids were produced as the changes were at different positions in the codon. Two mutations were detected at position 41 and mutations that occurred once were found at codons 69 and 80. Thus, 15 of the 25 second-site mutants were localized to codons 34 to 80 of VP5, which contains 1374 amino acids. The remaining 10 revertants had codon changes at nine different sites, of which the most N-terminal was altered at codon 187 and the most C-terminal at codon 1317. As noted in the much smaller study a preponderance of the second-site mutants in VP5 were altered in codons at the extreme N-terminus of VP5. It is especially noteworthy that 11 out of 25 of the mutations occurred at codons 34 and 78. As expected, all of the revertants isolated were shown to retain the original KUL26-610/611 mutation, and the scaffold proteins remain uncleaved. All showed decreased retention of VP24 in the B capsids compared to the wild-type KOS, but more than the KUL26-610/611 parental virus. The revertants all had decreased growth rates of 2 to 18% compared to that of KOS and showed varying degrees of sensitivity when grown at 39.5 degrees C. The mutations in VP5 of three of the previously isolated viruses (PR5, PR6, and PR7) were transferred into a wild-type background, i.e., a virus encoding wild-type UL26 and UL26.5 gene products. All replicated in nonpermissive (Vero) cells and cleaved scaffold proteins. PR5 and PR6 in the wild-type background gave wild-type burst sizes and gave C-capsids that retained VP24 at approximately wild-type levels. The third revertant, PR7, in the wild-type background showed only a twofold increase of burst size (to 20% of wild-type) and the capsids showed little or no increase of VP24 retention. Therefore, the second-site mutations of PR7 (R69C) by itself had a negative effect on virus replication. By contrast the temperature sensitivity of PR6 and PR7 remained unchanged in the wild-type background. Thus the temperature sensitivity of PR6 and PR7 resides in VP5 independently of the mutation in the UL26 cleavage site.

Relationship of hospital teaching status with quality of care and mortality for Medicare patients with acute MI.

CONTEXT: Issues of cost and quality are gaining importance in the delivery of medical care, and whether quality of care is better in teaching vs nonteaching hospitals is an essential question in this current national debate. OBJECTIVE: To examine the association of hospital teaching status with quality of care and mortality for fee-for-service Medicare patients with acute myocardial infarction (AMI). DESIGN, SETTING, AND PATIENTS: Analysis of Cooperative Cardiovascular Project data for 114,411 Medicare patients from 4361 hospitals (22,354 patients from 439 major teaching hospitals, 22,493 patients from 455 minor teaching hospitals, and 69,564 patients from 3467 nonteaching hospitals) who had AMI between February 1994 and July 1995. MAIN OUTCOME MEASURES: Administration of reperfusion therapy on admission, aspirin during hospitalization, and beta-blockers and angiotensin-converting enzyme inhibitors at discharge for patients meeting strict inclusion criteria; mortality at 30, 60, and 90 days and 2 years after admission. RESULTS: Among major teaching, minor teaching, and nonteaching hospitals, respectively, administration rates for aspirin were 91.2%, 86.4%, and 81.4% (P<.001); for angiotensin-converting enzyme inhibitors, 63. 7%, 60.0%, and 58.0% (P<.001); for beta-blockers, 48.8%, 40.3%, and 36.4% (P<.001); and for reperfusion therapy, 55.5%, 58.9%, and 55.2% (P =.29). Differences in unadjusted 30-day, 60-day, 90-day, and 2-year mortality among hospitals were significant at P<.001 for all time periods, with a gradient of increasing mortality from major teaching to minor teaching to nonteaching hospitals. Mortality differences were attenuated by adjustment for patient characteristics and were almost eliminated by additional adjustment for receipt of therapy. CONCLUSIONS: In this study of elderly patients with AMI, admission to a teaching hospital was associated with better quality of care based on 3 of 4 quality indicators and lower mortality. JAMA. 2000;284:1256-1262

Relation of race and sex to the use of reperfusion therapy in Medicare beneficiaries with acute myocardial infarction.

BACKGROUND: There are few reports describing the combined influence of the race and sex of a patient on the use of reperfusion therapy for acute myocardial infarction. METHODS: To determine the relation of race and sex to the receipt of reperfusion therapy for myocardial infarction in the United States, we reviewed the medical records of 234,769 Medicare patients with myocardial infarction. From these records we identified 26,575 white or black patients who met strict eligibility criteria for reperfusion therapy. We then performed bivariate and multivariate analyses of prevalence ratios to determine predictors of the use of reperfusion therapy in four subgroups of patients categorized according to race and sex: white men, white women, black men, and black women. RESULTS: Among eligible patients, white men received reperfusion therapy with the highest frequency (59 percent), followed by white women (56 percent), black men (50 percent), and black women (44 percent). After adjustment for differences in demographic and clinical characteristics, white women were as likely as white men to receive reperfusion therapy (prevalence ratio, 1.00; 95 percent confidence interval, 0.98 to 1.03). Likewise, black women were as likely as black men to receive reperfusion therapy (prevalence ratio, 1.00; 95 percent confidence interval, 0.89 to 1.13). However, black women were significantly less likely to receive reperfusion therapy than white men (prevalence ratio, 0.90; 95 percent confidence interval, 0.82 to 0.98), as were black men (prevalence ratio, 0.85; 95 percent confidence interval, 0.78 to 0.93). CONCLUSIONS: After adjustment for differences in clinical and demographic characteristics and clinical presentation, differences according to sex in the use of reperfusion therapy are minimal. However, blacks, regardless of sex, are significantly less likely than whites to receive this potentially lifesaving therapy.

Methods, results, and lessons learned from process evaluation of the high 5 school-based nutrition intervention.

This article describes the process evaluation of High 5, a school-based intervention targeting fruit and vegetable consumption among fourth graders and their families. The outcome evaluation involved 28 schools randomized to intervention or control conditions. The intervention included classroom, family, and cafeteria components. Process evaluation was completed on each of these components by using observations, self-report checklists, surveys, and other measures. Results indicated high implementation rates on the classroom activities. Moderate family involvement was attained, perhaps diminishing intervention effects on parent consumption. Cafeterias provided environmental cues, and fruit and vegetable offerings as directed by the program. A lower dose of the intervention was delivered to schools with larger African American enrollments and lower-income families. This article provides insights into the effective elements of a school-based dietary intervention and provides suggestions for process evaluation in similar studies.

Increasing the fruit and vegetable consumption of fourth-graders: results from the high 5 project.

BACKGROUND: This study evaluated the effects of a school-based dietary intervention program to increase fruit and vegetable consumption among fourth-graders. METHODS: Twenty-eight elementary schools were randomized to an immediate intervention condition or to a delayed intervention control condition. Measures of diet and psychosocial variables were collected at base line and 1 and 2 years post-baseline. The intervention included classroom, parent, and cafeteria components. RESULTS: Mean daily consumption of fruit and vegetables was higher for the intervention children compared with controls at Follow-up 1 (X(t) = 3.96, X(c) = 2.28) and at Follow-up 2 (X(t) = 3.20, X(c) = 2.21). Macro- and micronutrient changes favoring the intervention children were also observed at both Follow-up 1 and Follow-up 2. Mean daily consumption of fruit and vegetables was higher for intervention parents compared with controls at Follow-up 1 (X(t) = 4.23,X(c) = 3.94) but not at Follow-up 2. CONCLUSIONS: Strong effects were found for the High 5 intervention on fruit and vegetable consumption, on macro- and micro-nutrients, and on psychosocial variables. Future work is needed to enhance the intervention effects on parents' consumption and to test the effectiveness of the intervention when delivered by classroom teachers.

Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1.

VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.

Incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid.

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (Aequorea victoria) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.

Herpes simplex virus type 1 VP26 is not essential for replication in cell culture but influences production of infectious virus in the nervous system of infected mice.

VP26 is the smallest capsid protein of herpes simplex virus type 1 and is encoded by the UL35 open reading frame. It resides on the outer capsid surface, interacting with VP5 in a one to one stoichiometry in the hexons that comprise capsids. A null mutation in the gene encoding VP26 was generated and transferred into the KOS genome. Recombinant viruses were isolated on Vero cells, which indicated that the absence of VP26 was not required for growth of the virus in cell culture. This was confirmed by the characterization of the VP26 null mutant, designated K delta 26Z. The yield of virus from K delta 26Z-infected Vero cells was decreased only twofold relative to wild-type-infected cells, as judged by the burst size. All three types of capsids (A, B, and C) were observed after sedimentation analysis of K delta 26Z-infected cell extracts. These capsids were similar in composition to wild-type capsids except for the absence of VP26. The mouse ocular model was used to determine if VP26 played a major role in vivo. The yield of the mutant virus relative to wild-type virus was decreased twofold in the eye; however, the mutant virus yields were decreased 30- to 100-fold in the trigeminal ganglia. Reactivation of the mutant virus as determined by cocultivation assays was also reduced. To determine the effect of VP26 on capsid translocation, the VP26 null mutation was transferred into a virus specifiying a thymidine kinase mutation that by itself is transported to the trigeminal ganglia but whose DNA is not replicated in the ganglia. Using quantitative PCR assays the number of viral genomes detected in the ganglia was similar in the presence or the absence of VP26. Therefore, VP26 does not appear to aid in the translocation of the virus capsid from the mouse eye to the trigeminal ganglia but is important for infectious virus production in the ganglia.

Capsids are formed in a mutant virus blocked at the maturation site of the UL26 and UL26.5 open reading frames of herpes simplex virus type 1 but are not formed in a null mutant of UL38 (VP19C).

Previously we reported that null mutant viruses of UL19 (VP5) or of UL18 (VP23), essential components of herpes simplex virus type 1 (HSV-1) capsid shells, do not form precursor capsid structures as judged by sedimentation and electron microscope analysis. A goal of the present experiments was to isolate a null mutant virus for the remaining essential component of capsid shells, VP19C, encoded by the UL38 open reading frame (ORF). Furthermore, we wished to determine if a virus altered in the UL26 maturation cleavage site at residues 610 and 611 produced a lethal phenotype. Therefore, we decided to isolate cell lines that encode and express multiple capsid genes. Several cell lines were isolated by transformation of Vero cells and one designated C32 expressed all of the essential capsid proteins. Using this cell line we isolated a null mutant virus in the UL38 ORF and a mutant virus that was altered at residues 610 and 611 of the UL26 and UL26.5 gene products. We found that the null mutant in VP19C did not form a detectable product as judged by sedimentation and electron microscope analyses following infection of nonpermissive cells. The mutant virus altered at the UL26 maturation site resulted in the accumulation of B capsids. Therefore, cleavage at this site was essential for the maturation of B capsids into C capsids. Interestingly, the absence of cleavage at the maturation site was required for the retention of VP24 in the capsid.

The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA.

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.