In Situ TEM Study of the Degradation of PbSe Nanocrystals in Air

PbSe nanocrystals have attracted widespread attention due to a variety of potential applications. However, the practical utility of these nanocrystals has been hindered by their poor air stability, which induces undesired changes in the optical and electronic properties. An understanding of the degradation of PbSe nanocrystals when they are exposed to air is critical for improving the stability and enhancing their applications. Here, we use in situ transmission electron microscopy (TEM) with an environmental cell connected to air to study PbSe nanocrystal degradation triggered by air exposure. We have also conducted a series of complementary studies, including in situ environmental TEM study of PbSe nanocrystals exposed to pure oxygen and PbSe nanocrystals in H2O using a liquid cell, and ex situ experiments, such as O2 plasma treatment and thermal heating of PbSe nanocrystals under different air exposure. Our in situ observations reveal that when PbSe nanocrystals are exposed to air (or oxygen) under electron beam irradiation, they experience a series of changes, including shape evolution of individual nanocrystals with the cuboid intermediates, coalescence between nanocrystals, and formation of PbSe thin films through drastic solid-state fusion. Further studies show that the PbSe thin films transform into an amorphous Pb rich phase or eventually pure Pb, which suggest that Se reacts with oxygen and can be evaporated under electron beam illumination. These various in situ and ex situ experimental results indicate that PbSe nanocrystal degradation in air is initiated by the dissociation and removal of ligands from the PbSe nanocrystal surface

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

Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets

PAPP paralyzes <i>C</i>. <i>elegans</i> via SER-4 and DOP-3.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic non-NGM agar plates. <b>A.</b> PAPP (0.5 mM)-dependent paralysis of wild-type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for PAPP-dependent paralysis at 15 min exposure for wild type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). <b>C.</b> PAPP (0.5 mM)-dependent paralysis of 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing P<i>dop-3</i>::<i>dop-3</i> RNAi. Data are presented as mean ± SE (n = 3). ‘*’ p≤0.001, significantly different from 5-HT <i>quint</i> animals assayed under identical conditions.</p

5-HT and 5-HT receptor agonists selectively paralyze <i>C</i>. <i>elegans</i> 5-HT receptor mutant animals expressing nematode, insect or human 5-HT₁-like receptors in the cholinergic motor neurons.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. <b>A.</b> 5-HT (1 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> 8-OH-DPAT (2 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>C.</b> Sumatriptan (1 mM)-dependent paralysis of wild type, 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3).</p

<i>C</i>. <i>elegans</i> mutants with increased cuticular permeability are hypersensitive to 5-HT-dependent paralysis.

<p><b>A-B.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>A.</b> Wild type animals examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for 5-HT-dependent paralysis on NGM plates at 10 min exposure for wild type and 5-HT <i>quint</i> animals. <b>C-D.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on non-NGM agar (hypotonic) plates. <b>C.</b> Wild type animals were examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>D.</b> Dose-response curves for 5-HT-dependent paralysis in hypotonic conditions at 15 min exposure for wild type and 5-HT <i>quint</i> animals. <b>E-F.</b> 5-HT-dependent paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>E.</b> 5-HT (0.25 mM)-dependent paralysis of wild-type, <i>bus-8</i> (<i>e2968</i>), <i>bus-16</i> (<i>e2802</i>) and <i>bus-17</i> (<i>e2800</i>) mutants. Data are presented as mean ± SE (n = 3). <b>F.</b> Dose-response curves for 5-HT-dependent paralysis at 10 min exposure for wild type and <i>bus</i> mutants.</p

The 5-HT/SER-4-dependent inhibition of either the AIB interneurons or cholinergic motor neurons causes locomotory paralysis.

<p><b>A.</b> Confocal images of 5-HT <i>quint</i> expressing SER-4::GFP in the AIB interneurons (P<i>npr-9</i>)(A1) or cholinergic motor neurons (P<i>unc-17β</i>)(A2). GFP fluorescence (A2) or GFP fluorescence overlaid on DIC image (A1). The red stain in A2 is coelomocyte-specific RFP screening marker. <b>B.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. Wild type, quadruple 5-HT receptor null animals expressing only SER-4 (SER-4 <i>quad</i>) or 5-HT <i>quint</i> expressing the <i>C</i>. <i>elegans</i> 5-HT₁-like receptor, SER-4, in either the cholinergic motor neurons (P<i>unc-17β</i>) or the two AIB interneurons (P<i>npr-9</i>) were examined for 5-HT (1 mM)-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3).</p

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95high-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95