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Background:Failure to recognize children's overweight status by parents may contribute to children's risk for obesity. We examined two methods of measuring mothers' perceptions of children's weight and factors associated with weight perception inaccuracy.Methods:Cross-sectional analyses of clinical and self-report data from 287 Mexican-heritage mother–child dyads. Mothers identified their child's weight category using a scale (e.g., “normal/overweight/obese”) and a visual silhouette scale (11 child gender-specific weight-varying images). Children's height and weight were measured to calculate body mass index (BMI). Chi-square tests examined associations between categorical, silhouette, and BMI percentile categories of children's weight. Bivariate logistic regression analyses examined factors associated with mothers' inaccuracy of their children's weight.Results:Only 13% of mothers accurately classified their child as obese using the categorical scale, while 78% accurately classified their child as obese using the silhouette scale. Mothers were more likely to underestimate their child's weight using BMI categories (62%) compared to using the silhouette scale (23%). Predictors of mothers' underestimation using the categorical method were child sex [female] (adjusted odds ratio [AOR] = 1.99; 95% CI: 1.02–3.86), child age [younger age] (AOR = 10.39; 95% CI: 4.16–25.92 for ages 5–6 years), and mother's weight status (overweight AOR = 2.99; 95% CI: 1.05–8.51; obese AOR = 5.19; 95% CI: 1.89–14.18). Child BMI was the only predictor of mothers' overestimation (AOR = 0.89; 95% CI: 0.85–0.94) using the silhouette method.Conclusions:Using silhouette scales to identify children's body weight may be a more accurate tool for clinicians and interventionists to activate parents' awareness of unhealthy weight in children compared to using traditional categorical weight-labeling methods.

Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes.

Ophthalmic manifestations and tissue tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported in association with coronavirus disease 2019 (COVID-19), but the pathology and cellular localization of SARS-CoV-2 are not well characterized. The objective of this study was to evaluate macroscopic and microscopic changes and investigate cellular localization of SARS-CoV-2 across ocular tissues at autopsy. Ocular tissues were obtained from 25 patients with COVID-19 at autopsy. SARS-CoV-2 nucleocapsid gene RNA was previously quantified by droplet digital PCR from one eye. Herein, contralateral eyes from 21 patients were fixed in formalin and subject to histopathologic examination. Sections of the droplet digital PCR–positive eyes from four other patients were evaluated by in situhybridization to determine the cellular localization of SARS-CoV-2 spike gene RNA. Histopathologic abnormalities, including cytoid bodies, vascular changes, and retinal edema, with minimal or no inflammation in ocular tissues were observed in all 21 cases evaluated. In situhybridization localized SARS-CoV-2 RNA to neuronal cells of the retinal inner and outer layers, ganglion cells, corneal epithelia, scleral fibroblasts, and oligodendrocytes of the optic nerve. In conclusion, a range of common histopathologic alterations were identified within ocular tissue, and SARS-CoV-2 RNA was localized to multiple cell types. Further studies will be required to determine whether the alterations observed were caused by SARS-CoV-2 infection, the host immune response, and/or preexisting comorbidities.

AbstractBackground:Despite recent declines among young children, obesity remains a public health burden in the United States, including among Latino/Hispanic children. The determining factors are many and are too complex to fully address with interventions that focus on single factors, such as parenting behaviors or school policies. In this article, we describe a multisector, multilevel intervention to prevent and control childhood obesity in predominantly Mexican-origin communities in Southern California, one of three sites of the CDC-funded Childhood Obesity Research Demonstration (CA-CORD) study.Methods:CA-CORD is a partnership between a university-affiliated research institute, a federally qualified health center, and a county public health department. We used formative research, advisory committee members' recommendations, and previous research to inform the development of the CA-CORD project. Our theory-informed multisector, multilevel intervention targets improvements in four health behaviors: fruit, vegetable, and water consumption; physical activity; and quality sleep. Intervention partners include 1200 families, a federally qualified health center (including three clinics), 26 early care and education centers, two elementary school districts (and 20 elementary schools), three community recreation centers, and three restaurants. Intervention components in these sectors target changes in behaviors, policies, systems, and the social and physical environment. Evaluation activities include assessment of the primary outcome, BMI z-score, at baseline, 12-, and 18-months post-baseline, and sector evaluations at baseline, 12, and 24 months.Conclusions:Identifying feasible and effective strategies to prevent and control childhood obesity has the potential to effect real changes in children's current and future health status.

Purpose. To examine school socioeconomic status (SES) in relation to school physical activity-related practices and children's physical activity.Design. A cross-sectional design was used for this study.Setting. The study was set in 97 elementary schools (63% response rate) in two U.S. regions.Subjects. Of the children taking part in this study, 172 were aged 10.2 (standard deviation (SD) = 1.5) years; 51.7% were girls, and 69.2% were White non-Hispanic.Measures. School physical education (PE) teachers or principals responded to 15 yes/no questions on school physical activity-supportive practices. School SES (low, moderate, high) was derived from the percent of students eligible for free and reduced-price lunch. Children's moderate to vigorous physical activity (MVPA) during school was measured with accelerometers.Analysis. School level analyses involved linear and logistic regression; children's MVPA analyses used mixed effects regression.Results. Low-SES schools were less likely to have a PE teacher and had fewer physical activity-supportive PE practices than did high-SES schools (p < .05). Practices related to active travel to school were more favorable at low-SES schools (p < .05). Children attending high-SES schools had 4.4 minutes per day more of MVPA during school than did those at low-SES schools, but this finding was not statistically significant (p = .124).Conclusion. These findings suggest that more low- and moderate-SES elementary schools need PE teachers in order to reduce disparities in school physical activity opportunities and that PE time needs to be supplemented by classroom teachers or other staff to meet guidelines.

Previous research has been unable to identify a strong link between crude oil prices and economic news. We reexamine this relationship using high frequency intraday data and relatively new methodology to estimate jumps in oil prices. We find a surprisingly strong correspondence between high frequency jumps in oil prices and the arrival of new economic information, with the largest jumps tending to be preceded identifiable economic news. These results indicate that oil prices respond very rapidly to new economic data in ways that appear consistent with economic theory, and also suggest that economic news, rather than speculation unrelated to the economic environment, drives jumps in oil prices.

The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been cocrystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel inhibition of nucleation crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tmof apo-PR by 3.5–5 °C as assayed by differential scanning fluorimetry (DSF) and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.

AbstractBackground:Parents' employment status is frequently cited as a possible predictor of child weight status. Despite the importance of the topic, only a few studies have been conducted. No longitudinal studies have been conducted in the United States.Methods:A cohort of 1201 girls from the Trial of Activity for Adolescent Girls was used. Height, weight, and percent body fat (PBF) were measured at the 6thand 8thgrades. Parents' employment status (measured at 6thgrade) was categorized into working full time (reference), part time, unemployed, working or staying at home, and don't know. Mixed-model regression was used to reflect the hierarchical design of our study and adjusted for age, race, parents' education level, free or reduced-price school lunch status, and living arrangement.Results:Girls whose mothers worked part time or stayed at home had a decreased risk of excess weight gain [relative risk (RR)=0.94, 95% confidence interval (CI) 0.88, 1.00; RR=0.89, 95% CI 0.79, 1.00, respectively] compared to girls whose mothers worked full time. Girls whose fathers were unemployed had a moderately increased risk of excess weight gain (RR=1.13, 95% CI 1.00, 1.26) compared to girls whose fathers worked full time. Having an unemployed mother or part-time or stay-at-home father was not associated with excess weight gain. Parents' employment status was not associated with excess PBF gain.Conclusions:Our findings suggest that the availability of the mother has a greater influence on the weight of the daughter than the availability of the father. There is a need for a better understanding of how parents' employment status influences excess weight gain in adolescent girls.

Purpose. To examine pathways between individual, social, and environmental factors associated with leisure-time physical activity (LTPA) among Mexican-American adults.Design. Cross-sectional design using random-digit dialing to administer a structured telephone interview.Setting. Mexican-American adults living in a U.S.-Mexican border community in San Diego, California (N = 672).Measures. Data were collected on LTPA, demographic characteristics, acculturation, and other psychosocial and environmental factors associated with LTPA.Analysis. Structural equation modeling to test an a priori model of LTPA.Results. Participants were mostly female (71%) with a mean age of 39 years (SD = 13). Only 32% of participants met PA guidelines in their leisure time, with men (39%) meeting the guidelines more than women (29%). Using structural equation modeling, neighborhood factors, both social and environmental, showed indirect relationships with meeting PA guidelines through community resource factors. Significant covariates included marital status and age.Conclusion. Individual, social, and environmental factors were associated with LTPA in this sample of Mexican-American adults. These findings can inform intervention studies that aim to increase LTPA in this population.

AbstractBackground:This observational study examined cross-sectional and 24-month longitudinal associations of physical activity and dietary behaviors with change in BMI and percent body fat among children aged 6–9 years old.Methods:Data were from the control group (n=271; 48% Latino) of a community-based childhood obesity prevention program. Assessments were conducted at baseline and at 24 months and included height and weight, bioelectrical impedance–derived percent body fat, and 10 physical activity and dietary behaviors measured via parent report of their child. Cross-sectional analysis of variances (ANOVA) (normal weight, overweight, obese) and longitudinal mixed-effects linear regression models were used to investigate the relation of each physical activity and dietary behavior with BMI and percent body fat.Results:At baseline, obese children engaged in less physical activity and more sedentary behavior than normal-weight children (p<0.05). Increased physical activity (p<0.01) and number of breakfasts eaten with family (p<0.05) were associated with decreased BMI z-score and percent body fat. Decreased sedentary behavior and sugar-sweetened beverage consumption were associated with decreased percent body fat (p<0.05) but not BMI.Conclusions:In this cohort of 271 children, increased physical activity and eating breakfast with family and reduced screen-based sedentary behaviors and sugar-sweetened beverage consumption were associated with more favorable trends in adiposity. Therefore, attention to these behaviors may be of particular importance. Results also suggest that future studies should include percent body fat as an outcome for a more precise assessment of the association of behavior with adiposity.

Insecticide-treated nets (ITNs) reduce malaria transmission and related morbidity and child mortality; however, incorrect and inconsistent use limits their protective factors. This community trial titled the Net Use Intervention study sought to bridge the gap between ITN ownership and use in southern (coastal) Ghana and to determine the best mix of communication tools to affect behavior of ITN owners to consistent use while maintaining optimal internal and external validity. This two-group, non-randomized experiment evaluated a multichannel, multisector intervention process over the course of 8 weeks. A longitudinal cohort was scientifically sampled from six intervention and six control communities for both baseline and posttest surveys. The posttest survey showed no change in knowledge of ITNs in the intervention or control. In terms of use the previous night, there was a strong and statistically significant intervention effect (OR = 1.67; p< .05) within the intervention communities. The overall increase in ITN coverage was approximately one person per night per every two households. The promotion efforts succeeded well beyond the planners’ expectations, not only promoting usage but also dramatically increasing demand for new ITNs.

Insecticide-treated nets (ITNs) reduce malaria transmission and related morbidity and child mortality; however, incorrect and inconsistent use limits their protective factors. This community trial titled the Net Use Intervention study sought to bridge the gap between ITN ownership and use in southern (coastal) Ghana and to determine the best mix of communication tools to affect behavior of ITN owners to consistent use while maintaining optimal internal and external validity. This two-group, non-randomized experiment evaluated a multichannel, multisector intervention process over the course of 8 weeks. A longitudinal cohort was scientifically sampled from six intervention and six control communities for both baseline and posttest surveys. The posttest survey showed no change in knowledge of ITNs in the intervention or control. In terms of use the previous night, there was a strong and statistically significant intervention effect (OR = 1.67; p < .05) within the intervention communities. The overall increase in ITN coverage was approximately one person per night per every two households. The promotion efforts succeeded well beyond the planners' expectations, not only promoting usage but also dramatically increasing demand for new ITNs.

Latino children spend more time in sedentary activities than other American children, and only ~1 in 5 Latino children in public elementary and middle schools meet all 6 fitness standards in statewide fitness testing. Schools that facilitate physical activity (PA) by maintaining playgrounds and providing physical education classes have children who are more active and less overweight. The aims of the present study were to examine the extent to which several social and physical environmental changes in school settings resulted in observed changes in area characteristics and children's activity levels during recess. Thirteen elementary schools serving predominately Mexican American children were randomized into control or activity and nutrition environmental intervention conditions. Playgrounds and activities were restructured in 6 intervention schools to promote more PA. After 1 y, there were no overall statistical differences between treatment groups in PA or sedentary behavior in these settings and results did not differ by gender. Changing the social and physical environments to promote children's moderate-to-vigorous PA is important to the design of active and healthy recess environments. The present results are not conclusive as to the link between these interventions and actual behavior, but show sufficient promise for further population and setting specific research.

FIV is a significant pathogen in the cat and is, in addition, the smallest available natural model for the study of lentivirus infections. Although divergent at the amino acid level, the cat lentivirus has an abundance of structural and pathophysiological commonalities with HIV and thus serves well as a model for development of intervention strategies relevant to infection in both cats and man. The following review highlights both the strengths and shortcomings of the FIV/cat model, particular as regards development of antiviral drugs.

This paper extends the work in Serletis [Serletis, A. (1992). Unit root behavior in energy futures prices. The Energy Journal 13, 119–128] by re-examining the empirical evidence for random walk type behavior in energy futures prices. It tests for fractional integrating dynamics in energy futures markets utilizing more recent data (from January 3, 1994 to June 30, 2005) and a new semi-parametric wavelet-based estimator, which is superior to the more prevalent GPH estimator (on the basis of Monte-Carlo evidence). We find new evidence that energy prices display long memory and that the particular form of long memory is anti-persistence, characterized by the variance of each series being dominated by high frequency (low wavelet scale) components.

Purpose. This was a pilot study to assess the usability and impact of a feedback-based environmental tobacco smoke (ETS) reduction approach for Latino families.Design. A one-group, pre-post design was used.Methods. Information from hair samples from children analyzed for nicotine was used to create three feedback versions presented in a counterbalanced fashion. The intervention was conducted by a promotora in the participants' homes, with mailed materials and over the phone. Fifty Latino parent-child pairs were recruited. Parents were Spanish-speaking smoking adults (typically the mother) who reported exposing their children to ETS. Intervention efforts included home visits to provide nicotine feedback and counseling, two mailers presenting alternative versions of the feedback, and a supportive telephone call delivered over a 6-week period. Pre-post assessments conducted by a bicultural measurement technician measured parent reports of children's ETS exposure and children's hair nicotine. Parents' liking/usability of the three feedback formats was measured at the post-assessment. Paired t-tests assessed pre-post changes in exposure outcomes.Results. Parents' reports of exposure and children's hair nicotine levels showed statistically significant reductions. Parents liked all three feedback formats, particularly a comparative graphic format.Conclusions. Consistency of the positive findings suggests that this feedback approach to ETS reduction warrants further study with a more rigorous design.

Building from the results of a computational screen of a range of triazole-containing compounds for binding efficiency to human immunodeficiency virus type 1 protease (HIV-1-Pr), a novel series of potent inhibitors has been developed. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides ready access to 1,4-disubstituted-1,2,3-triazoles, was used to unite a focused library of azide-containing fragments with a diverse array of functionalized alkyne-containing building blocks. In combination with direct screening of the crude reaction products, this method led to the rapid identification of a lead structure and readily enabled optimization of both azide and alkyne fragments. Replacement of the triazole with a range of alternative linkers led to greatly reduced protease inhibition; however, further functionalization of the triazoles at the 5-position gave a series of compounds with increased activity, exhibiting Kivalues as low as 8 nM.

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The present study evaluated two relapse-prevention interventions intended to help women smokers maintain the "quit status" mandated during Navy basic training. Women with any smoking experience before entering the Navy (N = 2,781) were assigned to either a control group or to one of two intervention groups (access to a telephone help line or receive a series of monthly mailings) after graduating from basic training. Smoking/quit rates at 3, 6, and 12 months after graduation were assessed with mailed surveys. Response rates to the surveys ranged from 39% to 52%. There were no phone or mail intervention effects: cessation rates for the three conditions at the 12-month follow-up were 43% for control, 45% for the mail intervention, and 41% for the phone intervention. Results suggest the need for more aggressive relapse-prevention efforts, although quit rates were higher than expected "spontaneous" quit rates, indicating an impact of the totally smoke-free recruit training environment.

A global effort has been undertaken to control human immunodeficiency virus (HIV) though the development of vaccines and pharmacologics. Current FDA approved pharmacological inhibitors target two of the three viral enzymes critical to replication and maturation of infectious viral particles: reverse transcriptase (RT) and protease (Pr). Although combination therapies targeting RT and Pr have significantly reduced AIDS related morbidity and mortality, resistance to individual inhibitors is a growing concern. Currently, there are six protease inhibitors in clinical use. These inhibitors target the active site of protease using peptidomimetic transition state analogs based on natural substrates. However, treatment failures arise as a lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance. This allows reduced HIV-inhibitor pressure, increased viral replication, and the emergence of drug resistant mutations. Continued use of protease inhibitors in the face of incomplete viral suppression may result in HIV-1 escape mutants not only being resistant to the protease inhibitor used, but to all clinically available protease inhibitors. Thus, new broad-based protease inhibitors are needed to control the emerging multi-drug, cross-resistant HIV-1. Moreover, given the emergence of cross-resistant HIV-1, there is a need to target novel protease structural sites to reduce the risk of multi-drug cross-resistance. In this review, we discuss the resistance to protease inhibitors and the rationale for new strategies towards drug design for suppressing protease activity. We focus on the structure and function relationship and the influence that drug resistance mutants exert on the evolution of HIV-1 protease.

ABSTRACTThe calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-ß-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding—and the full complement of complex glycans—significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.

ABSTRACTThe feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning.

Several authors tare postulated that managed care and other related changes in the U.S. health care system, perceived negatively by mature physicians. may contribute to their early retirement. This, in turn, may produce advene effects on the availability of medical care. The authors investigate this and other related issues through analysis of a unique data vet obtained from a survey of nearly four thousand physicians, that elicited responses to numerous factors potentially relevant to their retirement decisions. The empirical analysis reveals that while managed care is an important factor in the retirement decision of physicians, it does not necessarily lead to earlier retirement. Physicians rank other factors, related to financial and personal issues, as more important in their retirement decisions and the authors find that several of these factors significantly impact their expected retirement age. The results have several implications for health care managers relating to the retention or retirement of practicing physicians, successful planning, and seamless generation of income.

Several retroviruses, including equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV) encode dUTPase. The role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dUTPase in virulence and viral mutation rate. Overall, the results of these studies have indicated a central role for dUTPase in facilitating productive viral replication in non-dividing cells. The requirement for dUTPase in EIAV, which replicates exclusively in macrophages, may be the most stringent. Studies of dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major determinant of virulence. In contrast, FIV readily replicates in dividing cell populations such as CD4+ and CD8+ T cells, and B cells as well as in non-dividing macrophages. Thus, the virus burden and disease sequelae are lowered in cats infected with a dUTPase-minus FIV relative to cats infected with wild type FIV, but not totally abrogated. Growth in macrophages is attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G--A transition mutations in viral integrants present in macrophages. These findings are consistent with an increase in uracil misincorporation in the absence of dUTPase, resulting in transition mutations that cripple the virus. Effects on virus replication and disease production have also been noted for dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase some reports suggest that other viral and host cell factors may substitute for its activity. Betaretroviruses also encode dUTPase and while several of these cause significant disease, the role of dUTPase in their replication and pathogenesis is currently unknown.

ABSTRACTTL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

ABSTRACTWe have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF?VVNGLVK-NH2(arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF?VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF?(CH2NH)VVNGL-NH2.This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.

Background.Each year, millions of children visit zoological parks, where they are exposed to long bouts of ultraviolet radiation (UVR). We conducted a study in the winter and replicated it in the summer to evaluate an intervention for reducing UVR exposure during the zoo visit.

ABSTRACTTo address the role of CXCR4 in the cell-surface attachment of the feline immunodeficency virus (FIV), a soluble fusion protein, gp95-Fc, consisting of the surface glycoprotein (SU, gp95) of either a primary (PPR) or cell line-adapted (34TF10) FIV strain was fused in frame with the Fc domain of human immunoglobulin G1. The recombinant SU-immunoadhesins were used as probes to investigate the cellular binding of FIV SU. In agreement with the host cell range properties of both viruses, binding of 34TF10 gp95-Fc was observed for all cell lines tested, whereas PPR gp95-Fc bound only to primary feline T cells. 34TF10 gp95-Fc also bound to Jurkat and HeLa cells, consistent with the ability of FIV to use human CXCR4 as a fusion receptor. As expected, 34TF10 gp95-Fc binding to Jurkat cells was blocked by addition of stromal cell-derived factor 1a (SDF-1a), as was binding to the 3201 feline lymphoma cell line. However, SDF-1a, RANTES, macrophage inflammatory protein 1ß, and heparin all failed to inhibit the binding of either gp95-Fc to primary T cells, suggesting that a non-CXCR4 receptor is involved in the binding of FIV SU. In this regard, an unidentified 40-kDa protein species from the surface of primary T cells but not Jurkat and 3201 cells specifically coprecipitated with both gp95-Fc. Yet another type of binding of 34TF10 gp95-Fc to adherent kidney cells was noted. SDF-1a failed to block the binding of 34TF10 gp95-Fc to either HeLa, Crandel feline leukemia, or G355-5 cells. However, binding was severely impaired in the presence of soluble heparin, as well as after enzymatic removal of surface heparans or on cells deficient in heparan expression. These overall findings suggest that in addition to CXCR4, a non-CXCR4 receptor and cell-surface heparans also play an important role in FIV gp95 cell surface interactions on specific target cells.

A series of norstatine-based HIV/FIV protease inhibitors incorporating a 15-membered macrocycle as a mimic of the tri-peptide (Ala-Val-Phe), a motif with a small P3′ residue effective against the FIV protease and the drug-resistant HIV proteases, has been synthesized. It was found that the macrocycle is important to the overall activity of the inhibitors. Certain inhibitors were developed expressing low nanomolar inhibitory activity against the HIV/FIV proteases and they are also effective against some drug-resistant as well as TL3-resistant HIV proteases.