RE-AIM Analysis of a School-Based Nutrition Education Intervention in Kindergarteners.

BACKGROUND: Few nutrition interventions in kindergarten classes have been evaluated, and none has been tested for program effectiveness, implementation, and dissemination. Building a Healthy Me (BHM) is a nutrition intervention for kindergarteners that is classroom-based and includes a family component. This study evaluated the public health impact of BHM in California kindergarten classrooms using the RE-AIM (reach, efficacy, adoption, implementation, and maintenance) framework. METHODS: A quasi-experimental design assessed pre-to-post changes in nutrition knowledge, dietary consumption, and parent behaviors of 25 intervention classrooms (414 students, 264 parents); and postintervention differences in nutrition knowledge between the intervention classrooms and 4 control classrooms measured at postintervention only (103 students). RESULTS: Intervention students improved in knowledge of food groups and healthy breakfast/snack options, and scored higher than control students in food group knowledge at postintervention (ps < .05). Parents of intervention group children increased their use of food labels, and intervention group children increased intake of several healthy foods and decreased intake of candy and fried potatoes (ps < .05). The BHM program reached 41% of kindergarteners attending public schools in California, and teachers implemented most lesson material. CONCLUSIONS: The BHM program was effective, implemented with fidelity, and broadly disseminated, highlighting its potential public health impact for kindergarteners.

FANCD2 limits BLM-dependent telomere instability in the alternative lengthening of telomeres pathway.

Fanconi anemia and Bloom syndrome are genomic instability syndromes caused by mutations in proteins that participate in overlapping DNA repair and replication pathways. Here, we show that the monoubiquitinated form of the Fanconi Anemia protein FANCD2 acts in opposition to the BLM DNA helicase to restrain telomere replication and recombination in human cells that utilize the Alternative Lengthening of Telomeres (ALT) pathway. ALT relies on exchanges of telomeric DNA to maintain telomeres, a process that we show FANCD2 suppresses. Depletion of FANCD2 results in a hyper-ALT phenotype, including an increase in extrachromosomal telomeric repeat DNAs, putative recombinational byproducts that we show exist as intertwined complexes forming the nucleic acid component of ALT-associated PML bodies. Increases in telomeric DNA are suppressed by loss of BLM but not RAD51, occur without parallel upregulation of shelterin proteins TRF1 and TRF2, and are associated with increased frequencies of deprotected and fragile telomeres. Inactivation of the FA pathway does not trigger ALT, as FANCD2 depleted telomerase positive cells do not acquire ALT-like phenotypes. We observe frequent fragile telomeres in ALT cells, suggesting that telomere sequences are prone to replication problems. We propose that, in ALT cells, FANCD2 promotes intramolecular resolution of stalled replication forks in telomeric DNA while BLM facilitates their resection and subsequent involvement in the intermolecular exchanges that drive ALT.

A general strategy for expanding polymerase function by droplet microfluidics.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

Comparative analysis of eukaryotic cell-free expression systems.

Cell-free protein synthesis (CFPS) allows researchers to rapidly generate functional proteins independent of cell culture. Although advances in eukaryotic lysates have increased the amount of protein that can be produced, the nuances of different translation systems lead to variability in protein production. To help overcome this problem, we have compared the relative yield and template requirements for three commonly used commercial cell-free translation systems: wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL). Our results provide a general guide for researchers interested in using cell-free translation to generate recombinant protein for biomedical applications.

RE-AIM analysis of a randomized school-based nutrition intervention among fourth-grade classrooms in California.

Childhood overweight and obesity are major health problems. School-based programs enable intervening with large groups of children, but program overall health impact is rarely completely assessed. A RE-AIM (Reach, Efficacy, Adoption, Implementation, Maintenance) analysis tested the overall public health impact of the fourth-grade "Nutrition Pathfinders" school-based nutrition-education program. A randomized controlled trial in 47 fourth-grade California classrooms (1713 students) tested program efficacy, and a secondary analysis of archival data tested program dissemination. Desired effects were seen in child nutrition knowledge, attitudes, consumption of low-nutrient high-density foods, sugar-sweetened beverages, proteins, grains, and parent willingness to serve new foods. The program was disseminated to ∼25 % of public school fourth-grade classrooms in California and cost about $1.00 per student to implement. The Nutrition Pathfinders program demonstrates potential for moderate to high public health impact due to its wide dissemination, effectiveness in altering attitudes and behaviors, and its relatively inexpensive cost of implementation.

DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.

Four Dietary Items of the School Physical Activity and Nutrition (SPAN) Questionnaire Form a Robust Latent Variable Measuring Healthy Eating Patterns.

OBJECTIVE: Evaluate the factor structure and stability of 4 dietary items (fruit, fruit juice, vegetables, and milk) from the School Physical Activity and Nutrition questionnaire-elementary school version. METHODS: Secondary analysis of intervention data from third graders measured at pre-intervention, post-intervention (10 weeks), and 3-month follow-up. The researchers conducted structural equation modeling invariance analysis to test the stability of the factor structure of the 4 items. RESULTS: Data from 1,147 students. Fit indices revealed good fit for a single factor remaining stable across time (χ(2)/degrees of freedom [DF] = 59.75/59, P = .45), gender (χ(2)/DF = 149.72/128, P = .09), and study groups (χ(2)/DF = 143.04/128, P = .17). CONCLUSIONS AND IMPLICATIONS: A healthy food factor consisting of the 4 items can be used in future data analysis. This offers several advantages in analysis, including the use of latent change scores that are more powerful, more informative, and more easily interpreted than traditional approaches.

Repurposing the antihelmintic mebendazole as a hedgehog inhibitor.

The hedgehog (Hh) signaling pathway is activated in many types of cancer and therefore presents an attractive target for new anticancer agents. Here, we show that mebendazole, a benzamidazole with a long history of safe use against nematode infestations and hydatid disease, potently inhibited Hh signaling and slowed the growth of Hh-driven human medulloblastoma cells at clinically attainable concentrations. As an antiparasitic, mebendazole avidly binds nematode tubulin and causes inhibition of intestinal microtubule synthesis. In human cells, mebendazole suppressed the formation of the primary cilium, a microtubule-based organelle that functions as a signaling hub for Hh pathway activation. The inhibition of Hh signaling by mebendazole was unaffected by mutants in the gene that encodes human Smoothened (SMO), which are selectively propagated in cell clones that survive treatment with the Hh inhibitor vismodegib. Combination of vismodegib and mebendazole resulted in additive Hh signaling inhibition. Because mebendazole can be safely administered to adults and children at high doses over extended time periods, we propose that mebendazole could be rapidly repurposed and clinically tested as a prospective therapeutic agent for many tumors that are dependent on Hh signaling.

Nutrition self-efficacy is unidirectionally related to outcome expectations in children.

OBJECTIVE: To clarify the underlying relationship between nutrition self-efficacy and outcome expectations because the direction of the relationship (unidirectional vs bidirectional) is debated in the literature. METHODS: Secondary data analysis of a 10-week, 10-lesson school-based nutrition education intervention among 3rd grade students (N = 952). Nutrition self-efficacy (7 items) and nutrition outcome expectations (9 items) were measured through student self-report at intervention pre- (time 1) and post- (time 2) assessments. A series of two time point, multi-group cross-lagged bivariate change score models were used to determine the direction of the relationship. RESULTS: A cross lag from nutrition self-efficacy at time 1 predicting changes in nutrition outcome expectations at time 2 significantly improved the fit of the model (Model 3), whereas a cross lag from nutrition outcome expectations at time 1 to changes in nutrition self-efficacy at time 2 only slightly improved the fit of the model (Model 2). Furthermore, adding both cross lags (Model 4) did not improve model fit compared to the model with only the self-efficacy cross lag (Model 3). Lastly, the nutrition outcome expectations cross lag did not significantly predict changes in nutrition self-efficacy in any of the models. CONCLUSIONS: Data suggest that there is a unidirectional relationship between nutrition self-efficacy and outcome expectations, in which self-efficacy predicts outcome expectations. Therefore, theory-based nutrition interventions may consider focusing more resources on changing self-efficacy because it may also lead to changes in outcome expectations as well.

Automated solid-phase synthesis of high capacity oligo-dT cellulose for affinity purification of poly-A tagged biomolecules.

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA-peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued. To overcome this problem, we have developed a low cost solid-phase synthesis protocol to generate oligo-dT cellulose. Comparative binding studies indicate that chemically synthesized oligo-dT cellulose functions with superior loading capacity when compared to the discontinued product. We suggest that this method could be used to synthesize oligo-dT resin for routine purification of poly-adenylated biomolecules.

A PTCH1 homolog transcriptionally activated by p53 suppresses Hedgehog signaling.

The p53-mediated responses to DNA damage and the Hedgehog (Hh) signaling pathway are each recurrently dysregulated in many types of human cancer. Here we describe PTCH53, a p53 target gene that is homologous to the tumor suppressor gene PTCH1 and can function as a repressor of Hh pathway activation. PTCH53 (previously designated PTCHD4) was highly responsive to p53 in vitro and was among a small number of genes that were consistently expressed at reduced levels in diverse TP53 mutant cell lines and human tumors. Increased expression of PTCH53 inhibited canonical Hh signaling by the G protein-coupled receptor SMO. PTCH53 thus delineates a novel, inducible pathway by which p53 can repress tumorigenic Hh signals.

General approach for characterizing in vitro selected peptides with protein binding affinity.

In vitro selection technologies are important tools for identifying high affinity peptides to proteins of broad medical and biological interest. However, the technological advances that have made it possible to generate long lists of candidate peptides have far outpaced our ability to characterize the binding properties of individual peptides. Here, we describe a low cost strategy to rapidly synthesize, purify, screen, and characterize peptides for high binding affinity. Peptides are assayed in a 96-well dot blot apparatus using membranes that enable partitioning of bound and unbound peptide-protein complexes. We have validated the binding affinity constants produced by this method using known peptide ligands and applied this process to discover five new peptides with nanomolar affinity to human α-thrombin. Given the need for new analytical tools that can accelerate peptide discovery and characterization, we feel that this approach would be useful to a wide range of technologies that utilize high affinity peptides.

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37-nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia-based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small-scale expression in 10(6) cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells.

Genome-wide profiling of human cap-independent translation-enhancing elements.

We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

Potential influence of plant chemicals on infectivity of Batrachochytrium dendrobatidis.

We explored whether extracts of trees frequently found associated with amphibian habitats in Australia and Arizona, USA, may be inhibitory to the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has been associated with global amphibian declines. We used salamanders Ambystoma tigrinum as the model system. Salamanders acquired significantly lower loads of Bd when exposed on leaves and extracts from the river red gum Eucalyptus camaldulensis, and loads were also low in some animals exposed on extracts of 2 oak species, Quercus emoryi and Q. turbinella. Some previously infected salamanders had their pathogen loads reduced, and some were fully cured, by placing them in leaf extracts, although some animals also self cured when housed in water alone. A significant number of animals cured of Bd infections 6 mo earlier were found to be resistant to reinfection. These results suggest that plants associated with amphibian habitats should be taken into consideration when explaining the prevalence of Bd in these habitats and that some amphibians may acquire resistance to the fungus if previously cured.

Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS.

Two-component systems are widely used by bacteria to mediate adaptive responses to a variety of environmental stimuli. The CusR/CusS two-component system in Escherichia coli induces expression of genes involved in metal efflux under conditions of elevated Cu(I) and Ag(I) concentrations. As seen in most prototypical two-component systems, signal recognition and transmission is expected to occur by ligand binding in the periplasmic sensor domain of the histidine kinase CusS. Although discussed in the extant literature, little experimental evidence is available to establish the role of CusS in metal homeostasis. In this study, we show that the cusS gene is required for Cu(I) and Ag(I) resistance in E. coli and that CusS is linked to the expression of the cusCFBA genes. These results show a metal-dependent mechanism of CusS activation and suggest an absolute requirement for CusS in Cu(I)- and Ag(I)-dependent upregulation of cusCFBA expression in E. coli.

Effects of sucrose and sorbitol on cement-based stabilization/solidification of toxic metal waste.

The effects of sucrose or sorbitol addition on the hydration, unconfined compressive strength and leachability of Portland cement pastes containing 1% Pb and 1% Zn were studied as a function of time. Whereas Pb and Zn were found to shorten the time to achieve maximum hydration of Portland cement, the combination of these metals with 0.15 wt% sucrose or 0.40 wt% sorbitol retarded the setting of cement by at least 7 and 28 days, respectively, without affecting the strength at 56 days. The leachability of Pb and Zn evaluated by the TCLP 1311 protocol at 56 and 71 days was slightly reduced or unchanged by the addition of sucrose or sorbitol. SEM-EDS and XRD analyses revealed that ettringite precipitation was favored whereas the formation of CSH gel, which accounts for most of the strength of hydrated cement, was delayed in cement pastes containing both metals and sucrose or sorbitol. These results indicate that controlled additions of sucrose or sorbitol can add flexibility to the handling of cement-treated metal waste, particularly when it needs to be transported by truck or pipeline between the treatment plant and the disposal site, without affecting its long-term performance.

Bidentate NHC-based chiral ligands for efficient Cu-catalyzed enantioselective allylic alkylations: structure and activity of an air-stable chiral Cu complex.

Cu-catalyzed addition of alkylzinc reagents to a range of allylic phosphates is promoted efficiently and with high enantioselectivity to afford tertiary as well as quaternary carbon centers (up to 98% ee). Reactions proceed to completion with 0.5-5 mol % catalyst loading and are best promoted by commercially available CuCl2.2H2O. The X-ray structure of the chiral NHC-Ag(I) complex used in the study as well as that of a catalytically active NHC-Cu(II) complex are also reported; both complexes are air-stable and are formed in >/=95% isolated yield. The isolated Cu complex, which can be handled in air, is catalytically active. The present report provides the first precedent for efficient Cu-catalyzed allylic alkylations with chiral NHC ligands.

Acid-Base Adducts of Catalytically Active Titanium(IV) Lewis Acids.

A series of monomeric Lewis acid-base adducts of the Diels-Alder catalyst Ti(O-2,6-Me(2)C(6)H(3))(2)Cl(2) have been synthesized from bidentate diphosphines and diamines, Ti(O-2,6-Me(2)C(6)H(3))(2)Cl(2)L(2) (L(2) = dmpe, depe, dpeda, and dmeda). X-ray crystal structures of Ti(O-2,6-Me(2)C(6)H(3))(2)Cl(2)(dmpe) and Ti(O-2,6-Me(2)C(6)H(3))(2)Cl(2)(dpeda) establish a distorted octahedral coordination environment with trans-chloride ligands. Bidentate ligands that were also studied but did not form isolable complexes with the Ti(IV) Lewis acid include dppe, tmeda, and binam. Through pairwise exchange reactions a qualitative ranking of relative bidentate ligand binding strengths to the Lewis acid were obtained (dmeda >/= dpeda > dmpe >/= depe > tmeda > binam > dppe). The ranking is readily rationalized using hard-soft electronic arguments except for tmeda, which requires that unfavorable steric interactions be invoked.