Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

TENSCell: Imaging of Stretch-Activated Cells Reveals Divergent Nuclear Behavior and Tension.

Mechanisms of cellular and nuclear mechanosensation are unclear, partially because of a lack of methods that can reveal dynamic processes. Here, we present a new concept for a low-cost, three-dimensionally printed device that enables high-magnification imaging of cells during stretch. We observed that nuclei of mouse embryonic skin fibroblasts underwent rapid (within minutes) and divergent responses, characterized by nuclear area expansion during 5% strain but nuclear area shrinkage during 20% strain. Only responses to low strain were dependent on calcium signaling, whereas actin inhibition abrogated all nuclear responses and increased nuclear strain transfer and DNA damage. Imaging of actin dynamics during stretch revealed similar divergent trends, with F-actin shifting away from (5% strain) or toward (20% strain) the nuclear periphery. Our findings emphasize the importance of simultaneous stimulation and data acquisition to capture mechanosensitive responses and suggest that mechanical confinement of nuclei through actin may be a protective mechanism during high mechanical stretch or loading.

The potential for indirect effects between co-flowering plants via shared pollinators depends on resource abundance, accessibility and relatedness.

Co-flowering plant species commonly share flower visitors, and thus have the potential to influence each other's pollination. In this study we analysed 750 quantitative plant-pollinator networks from 28 studies representing diverse biomes worldwide. We show that the potential for one plant species to influence another indirectly via shared pollinators was greater for plants whose resources were more abundant (higher floral unit number and nectar sugar content) and more accessible. The potential indirect influence was also stronger between phylogenetically closer plant species and was independent of plant geographic origin (native vs. non-native). The positive effect of nectar sugar content and phylogenetic proximity was much more accentuated for bees than for other groups. Consequently, the impact of these factors depends on the pollination mode of plants, e.g. bee or fly pollinated. Our findings may help predict which plant species have the greatest importance in the functioning of plant-pollination networks.

Associations of Religious Service Attendance With Cognitive Function in Midlife: Findings From The CARDIA Study.

OBJECTIVES: Growing evidence suggests that religiosity is an important social determinant of health, including cognitive health. Yet most prior work focused on older adults or was conducted in racially and denominationally homogeneous regional samples. This study investigates the association of religious service attendance in midlife with cognitive function later in midlife. METHODS: Using data from the Coronary Artery Risk Development in Young Adults (CARDIA) study, a racially and geographically diverse prospective cohort study, we explored the association of religious service attendance in midlife with cognitive function 5 years later. Cognitive function was measured using four cognitive tests administered by CARDIA technicians. Multivariable linear regression was used for analyses. Primary analyses controlled for sociodemographics, physical health, depression, and prior religious involvement. Sensitivity analyses additionally controlled for baseline cognition and social support. RESULTS: Our study population included 2,716 participants (57.2% female, 44.9% Black, and mean age 50). In primary analyses, attending services more than weekly (compared to never) in midlife was associated with better global cognition (ß = 0.14 standard deviations, 95% [confidence interval] CI = 0.02, 0.26) and verbal memory (ß = 0.17 standard deviations, 95% CI = 0.04, 0.30), but not with processing speed (ß = 0.04 standard deviations, 95% CI = -0.08, 0.16). A reverse association was observed with executive function (ß = -0.16 standard deviations, 95% CI = -0.30, -0.02). Most findings persisted in analyses accounting for loss to follow-up via inverse probability weighting. DISCUSSION: Our findings suggest that frequent involvement in religious services at midlife is associated with better global cognition and verbal memory but worse executive function. There was no association with processing speed.

Validation of a geospatial aggregation method for congressional districts and other US administrative geographies.

Stakeholders need data on health and drivers of health parsed to the boundaries of essential policy-relevant geographies. US Congressional Districts are an example of a policy-relevant geography which generally lack health data. One strategy to generate Congressional District heath data metric estimates is to aggregate estimates from other geographies, for example, from counties or census tracts to Congressional Districts. Doing so requires several methodological decisions. We refine a method to aggregate health metric estimates from one geography to another, using a population weighted approach. The method's accuracy is evaluated by comparing three aggregated metric estimates to metric estimates from the US Census American Community Survey for the same years: Broadband Access, High School Completion, and Unemployment. We then conducted four sensitivity analyses testing: the effect of aggregating counts vs. percentages; impacts of component geography size and data missingness; and extent of population overlap between component and target geographies. Aggregated estimates were very similar to estimates for identical metrics drawn directly from the data source. Sensitivity analyses suggest the following best practices for Congressional district-based metrics: utilizing smaller, more plentiful geographies like census tracts as opposed to larger, less plentiful geographies like counties, despite potential for less stable estimates in smaller geographies; favoring geographies with higher percentage population overlap.

Histone 3 Methyltransferases Alter Melanoma Initiation and Progression Through Discrete Mechanisms.

Perturbations to the epigenome are known drivers of tumorigenesis. In melanoma, alterations in histone methyltransferases that catalyze methylation at histone 3 lysine 9 and histone 3 lysine 27-two sites of critical post-translational modification-have been reported. To study the function of these methyltransferases in melanoma, we engineered melanocytes to express histone 3 lysine-to-methionine mutations at lysine 9 and lysine 27, which are known to inhibit the activity of histone methyltransferases, in a zebrafish melanoma model. Using this system, we found that loss of histone 3 lysine 9 methylation dramatically suppressed melanoma formation and that inhibition of histone 3 lysine 9 methyltransferases in human melanoma cells increased innate immune response signatures. In contrast, loss of histone 3 lysine 27 methylation significantly accelerated melanoma formation. We identified FOXD1 as a top target of PRC2 that is silenced in melanocytes and found that aberrant overexpression of FOXD1 accelerated melanoma onset. Collectively, these data demonstrate how histone 3 lysine-to-methionine mutations can be used to uncover critical roles for methyltransferases.

Auricular acupressure and acupuncture as adjuncts for pain management during first trimester medication abortion: A randomized three-arm trial.

OBJECTIVES: To measure pain during first trimester medication abortion using auricular acupressure or auricular acupuncture as an adjunct to pain management. We measured anxiety as a secondary outcome. STUDY DESIGN: This randomized, double-blinded, 3-arm trial enrolled women seeking medication abortion with mifepristone and misoprostol. Participants received auricular acupressure, auricular acupuncture, or inert auricular placebo patches immediately after receiving mifepristone. In addition, all participants received ibuprofen to use at home as needed. The study started with 1:1:1 randomization, but later overenrolled into the acupressure group after retraining for greater fidelity to that intervention. Participants reported pain and anxiety using numeric rating scales via text message for 4 days, and using a visual analog scale at follow-up. Analyses compared median pain scores of those receiving acupressure, acupuncture, or placebo. RESULTS: We randomized 136 participants of whom 57 received acupressure, 40 received acupuncture, and 39 received placebo. Groups had similar baseline characteristics. One hundred thirty-two participants (97%) reported outcomes by text message and 120 (88%) completed a follow-up interview. For acupressure, acupuncture and placebo groups the median maximum pain scores reported via text message were 60.0, 75.0, and 55.0 (p = 0.38); median maximum pain scores reported at follow-up were 76.5, 60.0, and 71.0 (p = 0.97), respectively. Acupressure results were similar before and after retraining. Maximum anxiety scores reported via text message were 10.0, 45.0, and 30.0 (p = 0.57). Maximum anxiety scores reported at follow-up were 10.5, 20.0, and 13.0 (p = 0.59). CONCLUSIONS: Pain and anxiety during medication abortion were similar among women receiving acupressure, acupuncture or placebo. IMPLICATIONS: We found no benefit in administering auricular acupressure or auricular acupuncture during medication abortion. These modalities are intended to be simple to use, but perhaps the brief provider training for this study was insufficient. These modalities should not be used in clinical practice without further study.

Conhecimento e implementação das práticas integrativas e complementares pelos enfermeiros da atenção básica / Knowledge and implementation of integrative and complementary practices by primary care nurses / Conocimiento e implementación de las prácticas integrativas y complementarias por los enfermeros de la atención básica

Objetivo: Identificar o conhecimento e a aplicação das práticas integrativas e complementares pelos enfermeiros da atenção básica. Métodos: É uma pesquisa descritiva de abordagem qualitativa envolvendo 19 enfermeiros da atenção básica do município de Mossoró situado no estado do Rio Grande do Norte, aprovada pelo comitê de ética e pesquisa sob o parecer 2.113.411. Resultados: Identificou-se quatro categorias: conhecimento dos enfermeiros sobre práticas integrativas; a não implementação das práticas integrativas e complementares na assistência do enfermeiro; implementação das práticas integrativas e complementares na assistência do enfermeiro; e as práticas são importantes para o cuidado em saúde. Conclusão: O conhecimento sobre a politica nacional de práticas integrativas e complementares, a variabilidade e a finalidade de tais práticas ainda se apresenta limitado, a solução para o conhecimento e implementação deficitários seria o ensino, seja ele na graduação ou na vida profissional

Objective: The study's purpose has been to identify the understanding and implementation of integrative and complementary practices by primary care nurses. Methods: It is a descriptive research with a qualitative approach that counted with the participation of 19 primary care nurses. This study took place in the municipality of Mossoró located in the Rio Grande do Norte State, being approved by the Research Ethics Committee under the Legal Opinion No. 2.113.411. Results: The following four categories were identified: nurses' knowledge regarding integrative practices; failure to implement integrative and complementary practices in nursing care; implementation of integrative and complementary practices in nursing care; and, the professional practices are important for health care. Conclusion: The knowledge concerning the national policy for integrative and complementary practices, the variability and the purpose of such practices is still limited. Bearing this in mind, the solution for such understanding and deficient implementation would be through teaching, being it during either graduation or professional life

Objetivo: Identificar el conocimiento e implementación de las prácticas integrativas y complementarias por los enfermeros de la atención básica. Métodos: Es una investigación descriptiva de abordaje cualitativo involucrando a 19 enfermeras de la atención básica del municipio de Mossoró, localizado en el estado de Rio Grande do Norte, aprobado por el comité de ética e investigación bajo la opinión 2.111.411. Resultados: Se identificaron cuatro categorías: conocimiento de enfermeros sobre prácticas integrativas; la no implementación de prácticas integrativas y complementarias en cuidados de enfermería; implementación de prácticas integradoras y complementarias en cuidados; y prácticas de enfermería son importantes para la atención de salud. Conclusión: El conocimiento sobre la política nacional de prácticas integrativas y complementarias, la variabilidad y el propósito de las mismas todavía está limitado, de esa forma, la solución sería la enseñanza