Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels

Binary-induced collapse of a compact, collisionless cluster

We improve and extend Shapiro's model of a relativistic, compact object which is stable in isolation but is driven dynamically unstable by the tidal field of a binary companion. Our compact object consists of a dense swarm of test particles moving in randomly-oriented, initially circular, relativistic orbits about a nonrotating black hole. The binary companion is a distant, slowly inspiraling point mass. The tidal field of the companion is treated as a small perturbation on the background Schwarzschild geometry near the hole; the resulting metric is determined by solving the perturbation equations of Regge and Wheeler and Zerilli in the quasi-static limit. The perturbed spacetime supports Bekenstein's conjecture that the horizon area of a near-equilibrium black hole is an adiabatic invariant. We follow the evolution of the system and confirm that gravitational collapse can be induced in a compact collisionless cluster by the tidal field of a binary companion.Comment: 9 Latex pages, 14 postscript figure

Class II MHC Self-Antigen Presentation in Human B and T Lymphocytes

Human CD4[superscript +] T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4[superscript +] T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.National Institutes of Health (U.S.) (grant P01AI039671)National Institutes of Health (U.S.) (P01AI045757

Addressing climate change with behavioral science:A global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions' effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior-several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people's initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors.</p

Addressing climate change with behavioral science: a global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions’ effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior—several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people’s initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors

Addressing climate change with behavioral science:A global intervention tournament in 63 countries

Effectively reducing climate change requires marked, global behavior change. However, it is unclear which strategies are most likely to motivate people to change their climate beliefs and behaviors. Here, we tested 11 expert-crowdsourced interventions on four climate mitigation outcomes: beliefs, policy support, information sharing intention, and an effortful tree-planting behavioral task. Across 59,440 participants from 63 countries, the interventions' effectiveness was small, largely limited to nonclimate skeptics, and differed across outcomes: Beliefs were strengthened mostly by decreasing psychological distance (by 2.3%), policy support by writing a letter to a future-generation member (2.6%), information sharing by negative emotion induction (12.1%), and no intervention increased the more effortful behavior-several interventions even reduced tree planting. Last, the effects of each intervention differed depending on people's initial climate beliefs. These findings suggest that the impact of behavioral climate interventions varies across audiences and target behaviors.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Food Preferences and Coping Strategies among Diabetic and Nondiabetic Households Served by US Food Pantries

Limited access to healthy food caused by food insecurity makes diabetes mellitus (DM) self-management more challenging. Using data from Hunger in America 2014 (n = 60,122 US food pantry users), we sought to understand food preferences and coping strategy utilization (e.g. choosing between paying for food and medical care) among households seeking assistance from US food pantries with and without DM members. The prevalence of wanting and not obtaining fruits, vegetables, dairy, and protein was high among all households. After adjusting for sociodemographic characteristics, households with DM members were more likely to want and not obtain fruits, vegetables, and dairy, and were also more likely to use several coping strategies to increase food access, compared to households without DM members. These results highlight the high demand for healthy food items among clients from US food pantries, particularly among households with DM, as well as the extra burden DM may place on food insecure households

A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in &gt;6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had &gt;90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known