Evidence for an interaction between the Galactic Center clouds M0.10-0.08 and M0.11-0.11

We present high-resolution (~2-3"; ~0.1 pc) radio observations of the Galactic center cloud M0.10-0.08 using the Very Large Array at K and Ka band (~25 and 36 GHz). The M0.10-0.08 cloud is located in a complex environment near the Galactic center Radio Arc and the adjacent M0.11-0.11 molecular cloud. From our data, M0.10-0.08 appears to be a compact molecular cloud (~3 pc) that contains multiple compact molecular cores (5+; <0.4 pc). In this study we detect a total of 15 molecular transitions in M0.10-0.08 from the following molecules: NH3, HC3N, CH3OH, HC5N, CH3CN, and OCS. We have identified more than sixty 36 GHz CH3OH masers in M0.10-0.08 with brightness temperatures above 400 K and 31 maser candidates with temperatures between 100-400 K. We conduct a kinematic analysis of the gas using NH3 and detect multiple velocity components towards this region of the Galactic center. The bulk of the gas in this region has a velocity of 51.5 km/s (M0.10-0.08) with a lower velocity wing at 37.6 km/s. We also detect a relatively faint velocity component at 10.6 km/s that we attribute to being an extension of the M0.11-0.11 cloud. Analysis of the gas kinematics, combined with past X-ray fluorescence observations, suggests M0.10-0.08 and M0.11-0.11 are located in the same vicinity of the Galactic center and could be physically interacting.Comment: Accepted for publication in the Astrophysical Journa

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

Turbulence And Heating Of Molecular Clouds In The Galactic Center

Molecular gas temperatures in the Galactic Center have been shown to much higher than the gas temperatures of molecular clouds in the disk. These Galactic Center clouds also show large line widths characteristic of turbulence. However, the origin of this heating and turbulence is not well known. In order to investigate this question we analyzed two Galctic Center molecular clouds that showed these characteristic: the G0.10-0.08 cloud and the M0.25+0.01 cloud. We observed these clouds using the VLA at K (25 GHz) and Ka (36 GHz) bands, both of which contain multiple molecular transitions including NH$_{3}$, CH$_{3}$OH and HC$_{3}$N. Using multiple transitions of NH$_{3}$, we determined that the rotational gas temperature in the clouds was $\sim$90-100 K. We also discovered multiple 36 GHz CH$_{3}$OH class I masers in both the G0.10-0.08 and M0.25+0.01 clouds, $\sim$50 and $\sim$80 respectively. Since these masers trace shocked gas, this indicates that some of this heating and turbulence is caused by these strong shocks. We also present images of the HC$_{3}$N line which is a high density tracer and shows dense cores at the center of both clouds

<i>Pitx1</i> determines nodule morphology <i>in vitro</i>.

<p><b>A–C;</b> Representative black and white images of 11.5dpc control forelimb (<b>A</b>) and hindlimb (<b>B</b>) and <i>Pitx1</i>^<i>-/-</i> hindlimb (<b>C</b>) micromasses stained for Alcian blue. <b>D–F;</b> Quantification of samples represented by A–C. Chondrogenic nodules in micromasses established from control hindlimbs (<b>B</b>) have larger total area, are larger in size (both measured by number of pixels) and have a larger hydraulic radius (<b>D–F</b>) than control forelimbs (<b>A</b>). In contrast, nodules in micromasses established from <i>Pitx1</i>^<i>-/-</i> hindlimbs (<b>C</b>) are significantly smaller, cover less total area, and have a smaller hydraulic radius than control hindlimb cultures (<b>B</b>, <b>D–F</b>) Control FL and HL; n = 6, <i>Pitx1</i>^<i>-/-</i> HL; n = 4. <b>G–H;</b> Micromasses generated from <i>Prx1-Pitx1</i>^<i>Tg/WT</i> forelimbs (<b>H</b>) which ectopically express <i>Pitx1</i> contain nodules which are larger and have a higher hydraulic radius, and cover a larger total area than control forelimb cultures (<b>G</b>, <b>I–K</b>). *p<0.05, **p<0.01, ***p<0.001, Student’s <i>t</i>-test.</p

Mouse forelimb and hindlimb micromass cultures display differences in patterning of chondrogenic nodules.

<p><b>A–B;</b> 17.5dpc mouse forelimb (<b>A</b>) and hindlimb (<b>B</b>) showing the three segments (stylopod, s, zeugopod, z and autopod, a) stained with Alcian blue for cartilage and Alizarin red for bone. Forelimb elements include the metacarpals (mc), and humerus (h). Hindlimb elements include the metatarsals (mt), the calcaneus (c), the patella (p) and the femur (f). <b>C–D;</b> A single nodule from a day 4 <i>Scleraxis-GFP</i> forelimb (<b>C</b>) and hindlimb (<b>D</b>) micromass showing tenoblasts (Scx-GFP; green), myoblasts (My32, red) and nuclei (DAPI, blue). <b>E–F;</b> Day 6 nodules present in forelimb (<b>E</b>) and hindlimb (<b>F</b>) micromasses display similar nodule makeup. Col2-GFP expression (green) is visible within cartilaginous nodules. <b>G–H;</b> Day 6 micromasses established from 11.5dpc mouse forelimb (<b>G</b>) and hindlimb (<b>H</b>) showing differences in size and shape of cartilage nodules stained by Alcian blue. <b>I–K;</b> Quantification of forelimb and hindlimb nodule characteristics across 6 experiments with >6 forelimb and hindlimb micromasses per experiment. Total chondrogenic area (<b>I</b>), nodule size (<b>J</b>) and hydraulic radius (area/perimeter; <b>K</b>) are larger in hindlimb cultures. P< 0.05, Student’s paired <i>t</i>-test. C–D; scale = 50μm.</p

Hindlimb cells are more adhesive <i>in vivo</i> and <i>in vitro</i> than forelimb cells.

<p><b>A</b>) Enzymatic dissociation of 11.5dpc limb buds with dispase releases more hindlimb cells than forelimb cells (p = 0.0021, n = 6). <b>B</b>) When micromass cultures are challenged after 1 hour of adhesion to the cell culture substrate, fewer hindlimb cells are left in the culture than forelimb cells (p = 0.011, n = 4 pools of >4 limbs each, normalised for limb number). *p<0.05, **p<0.01, ***p<0.001, Student’s <i>t</i>-test. <b>C–K;</b> Cell mixing experiments, 1:1 mix of <i>Prx1eGFP</i> hindlimb cells/wild type forelimb cells (<b>C–E</b>), <i>Prx1-eGFP</i> forelimb/wild type forelimb (<b>F–H</b>) and <i>Prx1-eGFP</i> hindlimb/wild type hindlimb (<b>I–K</b>) cells were stained for GFP protein (green) and nuclei (blue, DAPI).</p

<i>Pitx1</i> determines characteristic hindlimb morphologies in cartilage micromass culture

<div><p>The shapes of homologous skeletal elements in the vertebrate forelimb and hindlimb are distinct, with each element exquisitely adapted to their divergent functions. Many of the signals and signalling pathways responsible for patterning the developing limb bud are common to both forelimb and hindlimb. How disparate morphologies are generated from common signalling inputs during limb development remains poorly understood. We show that, similar to what has been shown in the chick, characteristic differences in mouse forelimb and hindlimb cartilage morphology are maintained when chondrogenesis proceeds <i>in vitro</i> away from the endogenous limb bud environment. Chondrogenic nodules that form in high-density micromass cultures derived from forelimb and hindlimb buds are consistently different in size and shape. We described analytical tools we have developed to quantify these differences in nodule morphology and demonstrate that characteristic hindlimb nodule morphology is lost in the absence of the hindlimb-restricted limb modifier gene <i>Pitx1</i>. Furthermore, we show that ectopic expression of <i>Pitx1</i> in the forelimb is sufficient to generate nodule patterns characteristic of the hindlimb. We also demonstrate that hindlimb cells are less adhesive to the tissue culture substrate and, within the limb environment, to the extracellular matrix and to each other. These results reveal autonomously programmed differences in forelimb and hindlimb cartilage precursors of the limb skeleton are controlled, at least in part, by <i>Pitx1</i> and suggest this has an important role in generating distinct limb-type morphologies. Our results demonstrate that the micromass culture system is ideally suited to study cues governing morphogenesis of limb skeletal elements in a simple and experimentally tractable <i>in vitro</i> system that reflects <i>in vivo</i> potential.</p></div

<i>Tbx5</i> Buffers Inherent Left/Right Asymmetry Ensuring Symmetric Forelimb Formation

<div><p>The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in <i>TBX5</i>, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor <i>Tbx5</i> in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of <i>Tbx5</i> produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In <i>Tbx</i> hypomorphic mutants maintained on an <i>INV</i> mutant background, with <i>situs inversus</i>, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of <i>Tbx5</i> are required to overcome this asymmetry to ensure symmetric forelimb formation.</p></div

<i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx;INV/INV</i> mutants with <i>situs inversus</i> have right biased asymmetric forelimb defects.

<p><b>A,</b><i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx-Tbx</i> mutant embryo at E14.5. The heart is on left (asterisk). <b>B,</b> Skeletal preparation. Right forelimb has 4 digits, while left forelimb has 3 digits (red arrow). <b>C,</b> <i>Tbx5</i>^{<i>lox/lox</i>}<i>;Prx1Cre;Prx1-Tbx;INV/INV</i> mutant embryo at E14.5. The heart is on the right (asterisk), indicating <i>situs inversus</i>. The right forelimb is more severely affected than the left (black arrow). <b>D,</b> Skeletal preparation. The left forelimb has 4 digits. In contrast, the right forelimb is more severely affected having 3 digits with the most anterior bifurcated (red arrow).</p