Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, making it difficult to understand how RNA structure governs function. Here we describe SHAPE mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2’-hydroxyl acylation by SHAPE are encoded as non-complementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure and was used to define a second-generation model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary structure models, enables analysis of low abundance RNAs, disentangles sequence polymorphisms in single experiments, and will ultimately democratize RNA structure analysis.

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This model is predictive of recent PrEP trial results in which 2-3 doses/week was 75%-90% effective in men but ineffective in women. These data provide a novel approach for future PrEP investigations that can optimize clinical trial dosing strategies.

Background Over 150 000 Malawians have started antiretroviral therapy (ART), in which first-line therapy is stavudine/lamivudine/nevirapine. We evaluated drug resistance patterns among patients failing first-line ART on the basis of clinical or immunological criteria in Lilongwe and Blantyre, Malawi. Methods Patients meeting the definition of ART failure (new or progressive stage 4 condition, CD4 cell count decline more than 30%, CD4 cell count less than that before treatment) from January 2006 to July 2007 were evaluated. Among those with HIV RNA of more than 1000 copies/ml, genotyping was performed. For complex genotype patterns, phenotyping was performed. Results Ninety-six confirmed ART failure patients were identified. Median (interquartile range) CD4 cell count, log10 HIV-1 RNA, and duration on ART were 68 cells/μl (23–174), 4.72 copies/ml (4.26–5.16), and 36.5 months (26.6–49.8), respectively. Ninety-three percent of samples had nonnucleoside reverse transcriptase inhibitor mutations, and 81% had the M184V mutation. The most frequent pattern included M184V and nonnucleoside reverse transcriptase inhibitor mutations along with at least one thymidine analog mutation (56%). Twenty-three percent of patients acquired the K70E or K65R mutations associated with tenofovir resistance; 17% of the patients had pan-nucleoside resistance that corresponded to K65R or K70E and additional resistance mutations, most commonly the 151 complex. Emergence of the K65R and K70E mutations was associated with CD4 cell count of less than 100 cells/μl (odds ratio 6.1) and inversely with the use of zidovudine (odds ratio 0.18). Phenotypic susceptibility data indicated that the nucleoside reverse transcriptase inhibitor backbone with the highest activity for subsequent therapy was zidovudine/lamivudine/tenofovir, followed by lamivudine/tenofovir, and then abacavir/didanosine. Conclusion When clinical and CD4 cell count criteria are used to monitor first-line ART failure, extensive nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor resistance emerges, with most patients having resistance profiles that markedly compromise the activity of second-line ART.

Twenty-two potential correlates of children's physical activity were examined. Two hundred and one Mexican-American and 146 Anglo-American families with 4-year-old children were studied. Children's physical activity was directly observed in the evening at home on 4 visits for 1 hr each time. Anglo-American children and male children were found to be more active. Demographic variables explained 11% of the variance in children's physical activity. After adjusting for demographics, 3 children's variables and 6 social-family variables did not account for significantly more variance. Five environmental variables accounted for 11% additional variance. Variables observed concurrently with physical activity, such as time spent outdoors and prompts to be active, were highly associated with children's physical activity.

An integrated system for coding direct observations of children's dietary and physical activity behaviors was developed. Associated environmental events were also coded, including physical location, antecedents, and consequences. To assess the instrument's reliability and validity, 42 children, aged 4 to 8 years, were observed for 8 consecutive weeks at home and at school. Results indicated that four 60-min observations at home produced relatively stable estimates for most of the 10 dimensions. Interobserver reliabilities during live and videotaped observations were high, with the exception of "consequences" categories that occurred in less than 1% of observed intervals. Evidence of validity was provided by findings that antecedents were associated with respective dietary and physical activity behaviors. The five physical activity categories were validated by heartrate monitoring in a second study. The Behaviors of Eating and Activity for Children's Health Evaluation System is appropriate for studying influences on diet and physical activity in children in a variety of settings.

Objective Examine the relationship between 1- and 2-month weight loss (WL) and 8-year WL among participants enrolled in a lifestyle intervention. Design & Methods 2290 Look AHEAD participants (BMI: 35.65±5.93kg/m2) with type 2 diabetes received an intensive behavioral WL intervention. Results 1 and 2-month WL were associated with yearly WL through Year 8 (p’s<0.0001). At Month 1, participants losing 2-4% and >4% had 1.62 (95% CI:1.32,1.98) and 2.79 (95% CI:2.21,3.52) times higher odds of achieving a ≥5% WL at Year 4 and 1.28 (95% CI:1.05,1.58) and 1.77 (95% CI:1.40,2.24) times higher odds of achieving a ≥5% at Year 8, compared to those losing <2% initially. At Month 2, a 3-6% WL resulted in greater odds of achieving a ≥5% WL at Year 4 (OR=1.85; CI:1.48,2.32) and a >6% WL resulted in the greatest odds of achieving a ≥5% WL at Year 4 (OR=3.85; CI:3.05,4.88) and Year 8 (OR=2.28; CI:1.81,2.89), compared to those losing <3%. Differences in adherence between WL categories were observed as early as Month 2. Conclusions 1 and 2-month WL was associated with 8-year WL. Future studies should examine whether alternative treatment strategies can be employed to improve treatment outcomes among those with low initial WL.

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