The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish.

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.The study was supported by Cancer Research UK (grant number C45041/A14953 to AC and LF), Wellcome Trust (grants number 084183/Z/07/Z to EBM and number 098051 to DLS and LLH), Specialist Programme from Bloodwise [12048], the Medical Research Council [MC_U105161083] and Ted’s Gang (to AJW), a Wellcome Trust strategic award to the Cambridge Institute for Medal Research [100140] and the Cambridge NIHR Biomedical Research Centre (to AJW and AC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It was first available from PLOS via http://dx.doi.org/10.1371/journal.pgen.100567

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

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

The number of HSPCs is rescued in a <i>tp53</i> mutant background.

<p>(A) The CHT of 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross stained by WISH against <i>c-myb</i>. Signal extracted from the corresponding WISH photograph is shown. Numbers represent larvae with the displayed phenotype out of the total number of larvae examined. (B) Quantification of <i>c-myb in situ</i> signal for 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross, depending on their genotype. Data are represented as the mean number of pixels +/- SEM. <i>nol9</i>^<i>+/+</i><i>;tp53</i>^<i>+/+</i> n = 9; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/+</i> n = 11; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^{<i>+/zdf1</i>} n = 19; <i>nol9</i>^<i>-/-</i><i>; tp53</i>^{<i>zdf1/zdf1</i>} n = 10. Two-tailed Student’s <i>t</i>-Test, *, p<0.05; **, p<0.01. (C) Representative pictures of the CHT of 96 hpf larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross stained by WISH against <i>hbae1</i>. Signal extracted from the corresponding WISH photograph is shown. (D) Quantification of <i>hbae1</i> WISH signal for larvae from a <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} x <i>nol9</i>^{<i>+/sa1022</i>}<i>;tp53</i>^{<i>+/zdf1</i>} cross, depending on their genotype. Data are represented as the mean number of pixels +/- SEM. <i>nol9</i>^<i>+/+</i><i>;tp53</i>^<i>+/+</i> n = 16; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/+</i> n = 14; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>+/-</i> n = 23; <i>nol9</i>^<i>-/-</i><i>;tp53</i>^<i>-/-</i> n = 16. Two-tailed Student’s <i>t</i>-Test, *, p<0.05; **, p<0.01. Within the figure, <i>nol9</i>^{<i>sa1022</i>} allele has been denoted as <i>nol9</i>^<i>-</i> and <i>tp53</i>^<i>zdf1</i> as <i>tp53</i>^<i>-</i>.</p

<i>nol9</i>^{<i>sa1022/sa1022</i>} embryos display tissue-specific upregulation of <i>tp53</i>.

<p>Representative images of embryos stained by whole-mount <i>in situ</i> hybridization against <i>tp53</i>. (A) At 48 hpf, similar levels of <i>tp53</i> signal (arrow) was detected in the CHT of <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos and their wild-type siblings. (B) At 72 hpf, <i>nol9</i>^{<i>sa1022/sa1022</i>} mutant embryos were characterized by more <i>tp53</i> signal in the CHT than their wt siblings. Mann-Whitney U test, p<0.05. (A-B) All embryos are oriented with anterior to the left and dorsal to the top. (C) Schematic representation of digestive organs in a wild-type 72 hpf zebrafish larva. I–intestine, L- liver, P- pancreas. (D) At 72 hpf, <i>nol9</i>^{<i>sa1022/sa1022</i>} mutant embryos display strong <i>tp53</i> signal in the liver (arrowhead) and intestine (arrow), compared to weak signal in the intestine of wild-type siblings. (C-D) Dorsal view anterior up.</p

Loss-of-function <i>nol9</i> mutation leads to a defect in ITS2 pre-rRNA processing.

<p>(A) Expression pattern of <i>nol9</i> by WISH at sphere stage (4 hpf), 12 hpf, 48 hpf, 72 hpf, 96 hpf and 120 hpf. Black arrows indicate branchial arches, white arrow indicates pancreas and arrowhead indicates <i>nol9</i>-expressing cells in the CHT. (B) Representative Northern blot analysis of RNA isolated from 5 dpf <i>nol9</i>^{<i>sa102/sa10222</i>} mutants and control (<i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>+/sa1022</i>}) siblings using 5’ETS, ITS1 and ITS2 probes to detect rRNA processing intermediates. Corresponding rRNA intermediates (a, b, c, d) are indicated in B) and C). (C) Schematic representation of the rRNA intermediates detected in the Northern blot analysis. The sites of hybridization of the 5’ETS, ITS1 and ITS2 probes are indicated in red. (D) Methylene blue staining of the membrane was used to control for equal loading of RNA. wt–<i>nol9</i>^<i>+/+</i>/<i>nol9</i>^{<i>+/sa1022</i>}, mut–<i>nol9</i>^{<i>sa1022/sa1022</i>}.</p

Ultrastructural studies of caudal hematopoietic tissue (CHT) in <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae.

<p>(A) Schematic representation of a transversal section in the CHT region of a 120 hpf zebrafish, dorsal up. NT–neural tube, NC–notochord, M—myotome, CA–caudal artery, CHT–caudal hematopoietic tissue, CV–caudal vein. (B-C) A low-magnification TEM image of the CHT from a <i>nol9</i>^<i>+/+</i> (B, n = 2) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (C, n = 2) larvae. Dashed line denotes the width of the CHT. Arrowheads denote mitochondrial profiles visible within myotomes (M). Asterisks denote extracellular matrix (ECM). (D-E) High magnification TEM image of cells present in the CHT of <i>nol9</i>^<i>+/+</i> (D) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (E) larvae. (F-G) High magnification TEM image of endothelial cells in the caudal vein of <i>nol9</i>^<i>+/+</i> (F) and <i>nol9</i>^{<i>sa1022/sa1022</i>} (G) larvae. Arrow denotes vesicles visible in the cytoplasm of the endothelial cell, reminiscent of lipofuscin. E–endothelium. Scale bars: 2 μm.</p

<i>nol9</i>^{<i>sa1022/sa1022</i>} mutants show a decrease in the number of lymphocytes and HSPCs.

<p>A) Whole-mount <i>in situ</i> hybridization with lymphocyte-specific <i>rag1</i> probe (arrowhead) revealed that <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae displayed weak to medium <i>rag1</i> expression at 96 hpf, compared to medium to strong signal in <i>nol9</i>^<i>+/+</i> or <i>nol9</i>^{<i>+/sa1022</i>} siblings. Larvae are oriented anterior to the top and ventral up. (B) The number of larvae displaying different degrees of <i>rag1</i> expression as assessed by WISH. Data are represented as the number of larvae belonging to each phenotypic group. Fisher’s exact test, ** p<0.01. (C) Whole-mount <i>in situ</i> hybridization against the thymic epithelium marker <i>foxn1</i> at 96 hpf revealed a similar level of signal (arrow) in <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} siblings. (D) Whole-mount <i>in situ</i> hybridization using a <i>c-myb</i> riboprobe was used to assess the number of HSCs emerging in the AGM region at 36 hpf (arrow) and the number of HSPCs in the CHT (arrow) at 72 hpf. Representative pictures of the AGM region (36 hpf) and the CHT (72 hpf) are shown. Mutant <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos displayed normal <i>c-myb</i> signal at 36 hpf and decreased <i>c-myb</i> signal at 72 hpf compared to wt siblings. Fisher’s exact test, **, p<0.01. All embryos are oriented with anterior to the left and dorsal to the top. Numbers represent embryos with the displayed phenotype out of the total number of embryos examined.</p

<i>nol9</i> mutation affects the development of the exocrine but not the endocrine pancreas.

<p>(A) Representative single channel confocal images of the pancreas of 96 hpf <i>Tg(ptf1a</i>:<i>EGFP;ins</i>:<i>mCherry)</i> larvae. <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae were characterized by smaller <i>ptf1a</i>-positive area (green) and similar <i>ins</i>-positive area (red) compared to <i>nol9</i>^<i>+/+</i> siblings. (B) TEM pictures of the exocrine pancreas of <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} larvae at 120 hpf. The white arrow denotes endoplasmic reticulum in the <i>nol9</i>^<i>+/+</i> cell. Asterisks indicate zymogen granules. Arrowheads indicate mitochondria. Scale bar: 2 μm. (C-D) Confocal images of the pancreas of <i>Tg(ptf1a</i>:<i>EGFP)</i> larvae subjected to immunohistochemistry against α-Carboxypeptidase-a (α-Cpa) (C) and α-Cytokeratin (D) at 120 hpf. (C) The exocrine pancreas differentiation marker α-Cpa (red) was detected in <i>nol9</i>^<i>+/+</i> and <i>nol9</i>^{<i>sa1022/sa1022</i>} siblings. (D) The pancreatic ducts expressing α-Cytokeratin (red) are not apparent in <i>nol9</i>^{<i>sa1022/sa1022</i>} mutants, in contrast to the ductal network clearly visible in <i>nol9</i>^<i>+/+</i> siblings.</p

<i>nol9</i>^{<i>sa1022/sa1022</i>} mutants show a decrease in proliferation of <i>c-myb</i>^<i>+</i> cells.

<p>(A) Representative maximum projection confocal images showing HSPCs (GFP^dim, arrow) in the CHT of <i>Tg(cd41</i>:<i>EGFP)</i> larvae at 96 hpf. Larvae are oriented with anterior to the left and dorsal to the top. (B) The number of GFP^dim HSPCs observed in the CHT region of <i>nol9</i>^{<i>sa1022/sa1022</i>} mutants (n = 20) and their wt siblings (n = 51) at 96 hpf. Data are represented as the mean +/- SEM, Student’s <i>t</i>-test, **, p<0.01. (C) Representative confocal images of the CHT of <i>Tg(cmyb</i>:<i>EGFP)</i> embryos subjected to BrdU incorporation assay at 48 hpf. Arrowheads mark double positive BrdU⁺ <i>c-myb</i>⁺ cells. Bright field image of a 48 hpf embryo shows the imaged part of the CHT. (D) The total number of <i>c-myb</i>^<i>+</i> cells observed in the CHT of <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos (n = 13) and their wt siblings (n = 14) subjected to the BrdU incorporation assay at 48 hpf. Data are represented as the mean +/- SEM. Student’s <i>t</i>-test. ns—not significant. (E) The percentage of BrdU⁺ cells within the <i>c-myb</i>^<i>+</i> population in the CHT of <i>nol9</i>^{<i>sa1022/sa1022</i>} embryos (n = 13) and their wt siblings (n = 14) subjected to the BrdU incorporation assay at 48 hpf. Data are represented as the mean +/- SEM, Student’s <i>t</i>-test, *, p<0.05.</p