RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using ...Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea
. Target interference relies on a multi-subunit, RNA-guided complex called Cascade
, which recruits a ...trans-acting helicase-nuclease, Cas3, for target degradation
. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain
and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.
Youth advocacy has been successfully used in substance use prevention but is a novel strategy in obesity prevention. As a precondition for building an evidence base for youth advocacy for obesity ...prevention, the present study aimed to develop and evaluate measures of youth advocacy mediator, process, and outcome variables.
The Youth Engagement and Action for Health (YEAH!) program (San Diego County, CA) engaged youth and adult group leaders in advocacy for school and neighborhood improvements to nutrition and physical activity environments. Based on a model of youth advocacy, scales were developed to assess mediators, intervention processes, and proximal outcomes of youth advocacy for obesity prevention. Youth (baseline n = 136) and adult group leaders (baseline n = 47) completed surveys before and after advocacy projects. With baseline data, we created youth advocacy and adult leadership subscales using confirmatory factor analysis (CFA) and described their psychometric properties.
Youth came from 21 groups, were ages 9-22, and most were female. Most youth were non-White, and the largest ethnic group was Hispanic/Latino (35.6%). The proposed factor structure held for most (14/20 youth and 1/2 adult) subscales. Modifications were necessary for 6 of the originally proposed 20 youth and 1 of the 2 adult multi-item subscales, which involved splitting larger subscales into two components and dropping low-performing items.
Internally consistent scales to assess mediators, intervention processes, and proximal outcomes of youth advocacy for obesity prevention were developed. The resulting scales can be used in future studies to evaluate youth advocacy programs.
As evidence grows about the benefits of policy and environmental changes to support active living and healthy eating, effective tools for implementing change must be developed. Youth advocacy, a ...successful strategy in the field of tobacco control, should be evaluated for its potential in the field of obesity prevention.
San Diego State University collaborated with the San Diego County Childhood Obesity Initiative to evaluate Youth Engagement and Action for Health! (YEAH!), a youth advocacy project to engage youth and adult mentors in advocating for neighborhood improvements in physical activity and healthy eating opportunities. Study objectives included documenting group process and success of groups in engaging in community advocacy with decision makers.
In 2011 and 2012, YEAH! group leaders were recruited from the San Diego County Childhood Obesity Initiative's half-day train-the-trainer seminars for adult leaders. Evaluators collected baseline and postproject survey data from youth participants and adult group leaders and interviewed decision makers.
Of the 21 groups formed, 20 completed the evaluation, conducted community assessments, and advocated with decision makers. Various types of decision makers were engaged, including school principals, food service personnel, city council members, and parks and recreation officials. Eleven groups reported change(s) implemented as a result of their advocacy, 4 groups reported changes pending, and 5 groups reported no change as a result of their efforts.
Even a brief training session, paired with a practical manual, technical assistance, and commitment of adult leaders and youth may successfully engage decision makers and, ultimately, bring about change.
Youth advocacy for obesity prevention is a promising but under-evaluated intervention. The aims of this study are to evaluate a youth advocacy program’s outcomes related to youth perceptions and ...behaviors, develop an index of youth advocacy readiness, and assess potential predictors of advocacy readiness. Youth ages 9–22 in an advocacy training program (
n
= 92 matched pairs) completed surveys before and after training. Youth outcomes and potential predictors of advocacy readiness were assessed with evaluated scales. All 20 groups who completed the evaluation study presented their advocacy projects to a decision maker. Two of six perception subscales increased following participation in the advocacy program: self-efficacy for advocacy behaviors (
p
< .001) and participation in advocacy (
p
< .01). Four of five knowledge and skills subscales increased: assertiveness (
p
< .01), health advocacy history (
p
< .001), knowledge of resources (
p
< .01), and social support for health behaviors (
p
< .001). Youth increased days of meeting physical activity recommendations (
p
< .05). In a mixed regression model, four subscales were associated with the advocacy readiness index: optimism for change (
B
= 1.46, 95 % CI = .49–2.44), sports and physical activity enjoyment (
B
= .55, 95 % CI = .05–1.05), roles and participation (
B
= 1.81, 95 % CI = .60–3.02), and advocacy activities (
B
= 1.49, 95 % CI = .64–2.32). The youth advocacy readiness index is a novel way to determine the effects of multiple correlates of advocacy readiness. Childhood obesity-related advocacy training appeared to improve youths’ readiness for advocacy and physical activity.
Cellular transformation induces phenotypically diverse populations of tumour-infiltrating T cells
, and immune checkpoint blockade therapies preferentially target T cells that recognize cancer cell ...neoantigens
. Yet, how other classes of tumour-infiltrating T cells contribute to cancer immunosurveillance remains elusive. Here, in a survey of T cells in mouse and human malignancies, we identified a population of αβ T cell receptor (TCR)-positive FCER1G-expressing innate-like T cells with high cytotoxic potential
(ILTCKs). These cells were broadly reactive to unmutated self-antigens, arose from distinct thymic progenitors following early encounter with cognate antigens, and were continuously replenished by thymic progenitors during tumour progression. Notably, expansion and effector differentiation of intratumoural ILTCKs depended on interleukin-15 (IL-15) expression in cancer cells, and inducible activation of IL-15 signalling in adoptively transferred ILTCK progenitors suppressed tumour growth. Thus, the antigen receptor self-reactivity, unique ontogeny, and distinct cancer cell-sensing mechanism distinguish ILTCKs from conventional cytotoxic T cells, and define a new class of tumour-elicited immune response.
Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found ...that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.
This article examines how midcentury marketing and manufacturing companies leveraged female designers in order to increase the consumption of their products. In 1956, the Aluminum Company of America ...(Alcoa) partnered with textile designer Marianne Strengell (1909-1989) to create a prototype of a nearly all aluminum rug for its Forecast advertising campaign. Facing stiff competition, Alcoa's goal was to expand the uses of its material beyond "pots and airplanes" by targeting women to buy home furnishings and clothing made of aluminum. Alcoa promoted the rug as well as Strengell in mainstream advertising seen by millions of Americans. By associating aluminum with soft fiber and women, Alcoa succeeded in broadening perceptions of aluminum beyond industrial applications.
We present a method to solve the problem of choosing a set of adverts to display to each of a sequence of web users. The objective is to maximise user clicks over time and to do so we must learn ...about the quality of each advert in an online manner by observing user clicks. We formulate the problem as a novel variant of a contextual combinatorial multi-armed bandit problem. The context takes the form of a probability distribution over the user's latent topic preference, and rewards are a particular nonlinear function of the selected set and the context. These features ensure that optimal sets of adverts are appropriately diverse. We give a flexible solution method which combines submodular optimisation with existing bandit index policies. User state uncertainty creates ambiguity in interpreting user feedback which prohibits exact Bayesian updating, but we give an approximate method that is shown to work well.
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