Community Recommendations for Improving Sustainable Scientific Software Practices

Multiple focus groups were conducted to elicit perspectives from members of the Earth science informatics community on the sustainability of scientific software. Recommendations that the participants offered for near-term community actions and activities are described

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

Novel Hypomorphic Alleles of the Mouse Tyrosinase Gene Induced by CRISPR-Cas9 Nucleases Cause Non-Albino Pigmentation Phenotypes

<div><p>Tyrosinase is a key enzyme in melanin biosynthesis. Mutations in the gene encoding tyrosinase (<i>Tyr</i>) cause oculocutaneous albinism (OCA1) in humans. Alleles of the <i>Tyr</i> gene have been useful in studying pigment biology and coat color formation. Over 100 different <i>Tyr</i> alleles have been reported in mice, of which ≈24% are spontaneous mutations, ≈60% are radiation-induced, and the remaining alleles were obtained by chemical mutagenesis and gene targeting. Therefore, most mutations were random and could not be predicted <i>a priori</i>. Using the CRISPR-Cas9 system, we targeted two distinct regions of exon 1 to induce pigmentation changes and used an <i>in vivo</i> visual phenotype along with heteroduplex mobility assays (HMA) as readouts of CRISPR-Cas9 activity. Most of the mutant alleles result in complete loss of tyrosinase activity leading to an albino phenotype. In this study, we describe two novel in-frame deletion alleles of <i>Tyr</i>, <i>dhoosara</i> (Sanskrit for gray) and <i>chandana</i> (Sanskrit for sandalwood). These alleles are hypomorphic and show lighter pigmentation phenotypes of the body and eyes. This study demonstrates the utility of CRISPR-Cas9 system in generating domain-specific in-frame deletions and helps gain further insights into structure-function of <i>Tyr</i> gene.</p></div

Comparison of <i>dhoosara</i> and <i>chandana</i> with the albino and black/wild type mice.

<p>(A) Dorsal view showing the coat color of albino, <i>chandana</i>, <i>dhoosara</i>, and black animals. Increasing levels of pigmentation can be seen in the hind limbs and tips of tails as well. (B) Frontal view of the face with increasing pigmentation in the nose region. Differences in the eye color can be noted. (C) Brightfield images of RPE wholemounts from Albino, <i>chandana</i>, <i>dhoosara</i> and Black mice. (D) Brightfield images of retinal cryosections from Albino, <i>chandana</i>, <i>dhoosara</i> and black mice. The RPE layer is the only layer within the eyeball that contains pigment. Scale bar = 50 μm. (E) Mean gray values (quantified in ImageJ) obtained from the RPE wholemounts reflecting relative intensity of RPE cell pigmentation. Results are presented in a bar chart with standard error of mean used for error bars. All groups are significantly different from each other (p<0.05, t-test). (F) Western Blot of tyrosinase protein isolated from skin of albino (alb), <i>chandana (cha)</i>, <i>dhoosara (dho)</i>, and black animals (arrow points to the 60 kDa tyrosinase band); 50 kDa tubulin protein (lower panel) is used as a control.</p

Details of the F0 animals obtained from CRISPR-Cas9 injections.

<p>Details of the F0 animals obtained from CRISPR-Cas9 injections.</p

CRISPR targeting and mutation detection by heteroduplex mobility assay in the <i>Tyr</i> gene.

<p>(A) Schematic showing CRISPR targeting regions (blue bold arrows), PCR primer binding sites, and amplicon sizes. (B) Breeding scheme with genotype of the zygotes used for CRISPR-Cas9 injections, and the CRISPR target sequences on both chromosomes. (C, D, E) Images of ethidium bromide stained polyacrylamide gels (6%) showing separation of homoduplex and heteroduplex PCR amplicons from CRISPR-Cas9 RNA injected, single cultured blastocysts (C, arrowheads) and tail DNA of potential founder mice (D, E). Gels on the left correspond to the 5’CRISPR target site, and those on the right correspond to the 3’CRISPR target site. Small and large square brackets indicate homoduplex and heteroduplex bands, respectively. L = 100 bp ladder; C = uninjected wildtype control.</p

Schematic of possible sequence of events producing related <i>dhoosara</i> and <i>chandana</i> alleles.

<p>CRISPR-Cas9 nuclease activity in the zygote at the 5’ target site on the wildtype chromosome results in a 15 bp deletion. After the cell division, nuclease activity persists and creates a DSB at the 3’ target site on the previously modified chromosome. NHEJ repair in this daughter cell results in a second, 3 bp deletion.</p

Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.

<p>Multiples mutant alleles resulting from NHEJ at 5’ and 3’ CRISPR target sites.</p

Political Accountability Under Alternative Institutional Regimes

This article contributes to the development of a positive theory of the interaction between institutional checks and balances and public accountability. In particular, the authors are interested in how various institutional separation-of-powers rules affect voter behavior, and in how these rational voter responses may affect our positive and normative assessment of different separation-of-powers regimes. The authors compare three stylized institutional arrangements: The first is a “Unilateral Authority”. The second regime is a “Mandatory Checks and Balances” regime. The third and final regime they consider is an “Opt-In Checks and Balances” system. These are obviously only three of a much larger number of possible institutional arrangements, but their simple structure is useful in generating comparative insights that might then be transposed to more complex and realistic institutional settings