Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

[EN] Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.A.A. and J.A.M. thank the European Syncrotron Radiation Facility and EMBL for access to the synchrotron radiation source. This work was funded by Ministerio de Economia y Competitividad (MINECO) Grants BFU2014-59796-R (to A.A.), BFU2011-28184-C02 (to M.J.S.-B.), and BIO2014-52537-R (to P.L.R.) and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A and M.M.). M.J.S.-B. is supported by Ramon y Cajal Contract RYC-2008-03449 from MINECO and M.D. by a fellowship from Senacyt-Ifarhu. Access to the High Throughput Crystallization facility at European Molecular Biology Laboratory (EMBL) Grenoble was supported by the European Community's Seventh Framework Programme through the Protein Production Platform project (P-CUBE) Grant 227764.Diaz, M.; Sanchez-Barrena, MJ.; Gonzalez Rubio, JM.; Rodríguez Solovey, LN.; Fernández, D.; Antoni-Alandes, R.; Yunta, C.... (2016). Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling. Proceedings of the National Academy of Sciences. 113(3):E396-E405. https://doi.org/10.1073/pnas.1512779113SE396E4051133Serrano, R., & Rodriguez-Navarro, A. (2001). 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PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms

[EN] The phytohormone abscisic acid (ABA) plays a key role regulating root growth, root system architecture, and root adaptive responses, such as hydrotropism. The molecular and cellular mechanisms that regulate the action of core ABA signaling components in roots are not fully understood. ABA is perceived through receptors from the PYR/PYL/RCAR family and PP2C coreceptors. PYL8/RCAR3 plays a nonredundant role in regulating primary and lateral root growth. Here we demonstrate that ABA specifically stabilizes PYL8 compared with other ABA receptors and induces accumulation of PYL8 in root nuclei. This requires ABA perception by PYL8 and leads to diminished ubiquitination of PYL8 in roots. The ABA agonist quinabactin, which promotes root ABA signaling through dimeric receptors, fails to stabilize the monomeric receptor PYL8. Moreover, a PYL8 mutant unable to bind ABA and inhibit PP2C is not stabilized by the ligand, whereas a PYL85KR mutant is more stable than PYL8 at endogenous ABA concentrations. The PYL8 transcript was detected in the epidermis and stele of the root meristem; however, the PYL8 protein was also detected in adjacent tissues. Expression of PYL8 driven by tissue-specific promoters revealed movement to adjacent tissues. Hence both inter- and intracellular trafficking of PYL8 appears to occur in the root apical meristem. Our findings reveal a non-cell-autonomous mechanism for hormone receptors and help explain the nonredundant role of PYL8-mediated root ABA signaling.Work in the P.L.R. and F.M. laboratories was supported by the Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas Grants BIO2014-52537-R and BIO2017-82503-R (to P.L.R.) and BIO2015-64307-R (to F.M.). J.L.-J. was supported by a Juan de la Cierva contract from Ministerio de Economia y Competitividad (MINECO) and by the Marie Sklodowska-Curie Action H2020-MSCA-IF-2015-707477. B.B.-P. was funded by Programa VALi+d GVA APOSTD/2017/039. J.J. was supported by a FPI contract from MINECO and M.A.F. by a Formacion de Profesorado Universitario contract from MINECO. D.D. and M.J.B. were supported by Biotechnology and Biological Sciences Research Council Grant BB/M002136/1 and Leverhulme Trust Grant RPG-2016-409.Belda-Palazón, B.; Gonzalez-Garcia, M.; Lozano Juste, J.; Coego Gonzalez, A.; Antoni-Alandes, R.; Julian-Valenzuela, J.; Peirats-Llobet, M.... (2018). PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms. Proceedings of the National Academy of Sciences of the United States of America (Online). 115(50):E11857-E11863. https://doi.org/10.1073/pnas.1815410115SE11857E1186311550Ubeda-Tomás, S., Beemster, G. T. S., & Bennett, M. J. 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Endodermal ABA Signaling Promotes Lateral Root Quiescence during Salt Stress in Arabidopsis Seedlings. The Plant Cell, 25(1), 324-341. doi:10.1105/tpc.112.107227Feng, W., Lindner, H., Robbins, N. E., & Dinneny, J. R. (2016). Growing Out of Stress: The Role of Cell- and Organ-Scale Growth Control in Plant Water-Stress Responses. The Plant Cell, 28(8), 1769-1782. doi:10.1105/tpc.16.00182Geng, Y., Wu, R., Wee, C. W., Xie, F., Wei, X., Chan, P. M. Y., … Dinneny, J. R. (2013). A Spatio-Temporal Understanding of Growth Regulation during the Salt Stress Response in Arabidopsis. The Plant Cell, 25(6), 2132-2154. doi:10.1105/tpc.113.112896Takahashi, N., Goto, N., Okada, K., & Takahashi, H. (2002). Hydrotropism in abscisic acid, wavy, and gravitropic mutants of Arabidopsis thaliana. Planta, 216(2), 203-211. doi:10.1007/s00425-002-0840-3Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Peirats-Llobet, M., Pizzio, G. A., Fernandez, M. A., … Rodriguez, P. L. (2012). PYRABACTIN RESISTANCE1-LIKE8 Plays an Important Role for the Regulation of Abscisic Acid Signaling in Root. Plant Physiology, 161(2), 931-941. doi:10.1104/pp.112.208678Barberon, M., Vermeer, J. E. M., De Bellis, D., Wang, P., Naseer, S., Andersen, T. G., … Geldner, N. (2016). Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation. Cell, 164(3), 447-459. doi:10.1016/j.cell.2015.12.021Ondzighi-Assoume, C. A., Chakraborty, S., & Harris, J. M. (2016). Environmental Nitrate Stimulates Abscisic Acid Accumulation in Arabidopsis Root Tips by Releasing It from Inactive Stores. The Plant Cell, 28(3), 729-745. doi:10.1105/tpc.15.00946Irigoyen, M. L., Iniesto, E., Rodriguez, L., Puga, M. I., Yanagawa, Y., Pick, E., … Rubio, V. (2014). Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis. The Plant Cell, 26(2), 712-728. doi:10.1105/tpc.113.122234Bueso, E., Rodriguez, L., Lorenzo-Orts, L., Gonzalez-Guzman, M., Sayas, E., Muñoz-Bertomeu, J., … Rodriguez, P. L. (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. The Plant Journal, 80(6), 1057-1071. doi:10.1111/tpj.12708Knoblich, J. A. (2005). Pins for spines. Nature Cell Biology, 7(12), 1057-1058. doi:10.1038/ncb1205-1057Zhang, H., Han, W., De Smet, I., Talboys, P., Loya, R., Hassan, A., … Wang, M.-H. (2010). ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem. The Plant Journal, 64(5), 764-774. doi:10.1111/j.1365-313x.2010.04367.xBelda-Palazon, B., Rodriguez, L., Fernandez, M. A., Castillo, M.-C., Anderson, E. M., Gao, C., … Rodriguez, P. L. (2016). 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C., Defries, A., Park, S.-Y., Endo, A., Nambara, E., … Cutler, S. R. (2013). Activation of dimeric ABA receptors elicits guard cell closure, ABA-regulated gene expression, and drought tolerance. Proceedings of the National Academy of Sciences, 110(29), 12132-12137. doi:10.1073/pnas.1305919110Cao, M., Liu, X., Zhang, Y., Xue, X., Zhou, X. E., Melcher, K., … Xu, Y. (2013). An ABA-mimicking ligand that reduces water loss and promotes drought resistance in plants. Cell Research, 23(8), 1043-1054. doi:10.1038/cr.2013.95Castillo, M.-C., Lozano-Juste, J., González-Guzmán, M., Rodriguez, L., Rodriguez, P. L., & León, J. (2015). Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants. Science Signaling, 8(392), ra89-ra89. doi:10.1126/scisignal.aaa7981Wu, S., & Gallagher, K. L. (2014). The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system. 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Chemical Genetics Reveals Negative Regulation of Abscisic Acid Signaling by a Plant Immune Response Pathway. Current Biology, 21(11), 990-997. doi:10.1016/j.cub.2011.04.045Waadt, R., Hitomi, K., Nishimura, N., Hitomi, C., Adams, S. R., Getzoff, E. D., & Schroeder, J. I. (2014). FRET-based reporters for the direct visualization of abscisic acid concentration changes and distribution in Arabidopsis. eLife, 3. doi:10.7554/elife.01739Jones, A. M., Danielson, J. Å., ManojKumar, S. N., Lanquar, V., Grossmann, G., & Frommer, W. B. (2014). Abscisic acid dynamics in roots detected with genetically encoded FRET sensors. eLife, 3. doi:10.7554/elife.01741Zhao, Y., Xing, L., Wang, X., Hou, Y.-J., Gao, J., Wang, P., … Zhu, J.-K. (2014). The ABA Receptor PYL8 Promotes Lateral Root Growth by Enhancing MYB77-Dependent Transcription of Auxin-Responsive Genes. Science Signaling, 7(328), ra53-ra53. doi:10.1126/scisignal.2005051Peirats-Llobet, M., Han, S.-K., Gonzalez-Guzman, M., Jeong, C. 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Preemptive-TIPS improves outcome in high-risk variceal bleeding : An observational study

Objective Patients admitted with acute variceal bleeding (AVB) and Child Pugh C score (CP\u2010C) or Child Pugh B plus active bleeding at endoscopy (CP\u2010B+AB) are at high risk for treatment failure, rebleeding and mortality. Preemptive TIPS (p\u2010TIPS) has been shown to improve survival in these patients but its use in clinical practice has been challenged and not routinely incorporated. The present study aimed to further validate the role of preemptive TIPS in a large number of high\u2010risk patients. Design Multicenter, international, observational study including 671 patients from 34 centers admitted for AVB and high\u2010risk of treatment failure. Patients were managed according to current guidelines and use of drugs and endoscopic therapy (D+E) or preemptive TIPS (p\u2010TIPS) was based on individual center policy. Results p\u2010TIPS in the setting of AVB is associated with a lower mortality in Child C patients compared to D+E (1 year mortality 22% vs 47% in D+E group; P=0.002). Mortality rate in CP\u2010B+AB patients was low and p\u2010TIPS did not improve it. In CP\u2010C and CP\u2010B +AB patients, p\u2010TIPS reduces treatment failure and rebleeding (1 year CIF\u2010probability of remaining free of the composite endpoint: 92% vs 74% in the D+E group; P=0.017), development of \u201cde novo\u201d or worsening of previous ascites without increasing rates of hepatic encephalopathy. Conclusion p\u2010TIPS must be the treatment of choice in CP\u2010C patients with AVB. Due to the strong benefit in preventing further bleeding and ascites, p\u2010TIPS could be a good treatment strategy for CP\u2010B+AB patients

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Climate Trends Impact on the Snowfall Regime in Mediterranean Mountain Areas : Future Scenario Assessment in Sierra Nevada (Spain)

Snow constitutes a key component of the water cycle, which is directly affected by changes in climate. Mountainous regions, especially those located in semiarid environments, are highly vulnerable to shifts from snowfall to rainfall. This study evaluates the influence of future climate scenarios on the snowfall regime in the Sierra Nevada Mountains, an Alpine/Mediterranean climate region in southern Spain. Precipitation and temperature projections from two future climate scenarios representative concentration pathway (RCP) 4.5 and RCP 8.5, Fifth Assessment Report of the Intergovernmental Panel for Climate Change (AR5 IPCC)) were used to estimate the projected evolution of the snowfall regime on both annual and decadal scales during the period of 2006&ndash;2100. Specific snowfall descriptors of torrentiality are also analyzed. A general decrease of the annual snowfall was estimated, with a significant trend that ranged from 0.21 to 0.55 (mm&middot;year&minus;1)&middot;year&minus;1. These changes are dependent on the scenario and region in the study area. However, the major impact of future climate scenarios on the snowfall regime relates to an increased torrentiality of snowfall occurrence, with a decreased trend of the annual number of snowfall days (RCP 4.5: &minus;0.068 (days&middot;year&minus;1)&middot;year&minus;1 and RCP 8.5: &minus;0.111 (days&middot;year&minus;1)&middot;year&minus;1) and an increased trend in the annual mean snowfall intensity (RCP 4.5: 0.006 (mm&middot;days&minus;1)&middot;year&minus;1 and RCP8.5: 0.01 (mm&middot;days&minus;1)&middot;year&minus;1)) under both scenarios. This enhanced torrentiality is heterogeneously distributed, with the most semiarid region, which is currently the one least influenced by snow, being the region most affected within the study area

Evaluation of the Diagnostic Capability of Spectralis SD-OCT 8 × 8 Posterior Pole Software with the Grid Tilted at 7 Degrees and Horizontalized in Glaucoma

Background: The goal was to evaluate the diagnostic capability of different parameters obtained with the posterior pole (PP) software in Spectralis SD-OCT with the 8 × 8 grid tilted at 7° and horizontalized in glaucomatous eyes. Methods: A total of 299 eyes were included, comprising 136 healthy eyes and 163 with primary open-angle glaucoma (POAG). The following segmentations were evaluated: complete retina, retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), GCL and inner plexiform layer (GCLIPL), ganglion cell complex (GCC), outer plexiform layer and outer nuclear layer (OPLONL), inner retinal layer (IRL), and outer retinal layer (ORL). Different patterns of macular damage were represented using heatmaps for each studied layer, where the areas under the curve (AUROC) values and a retinal thickness cutoff point were defined to discriminate POAG patients. Results: There was not any difference in the diagnostic capability for detecting glaucoma between the grid tilted at 7° and horizontalized. The macular segmentations that offer the highest diagnostic ability in glaucoma discrimination were, in the following order, RNFL (AUROC = 0.796), GCC (AUROC = 0.785), GCL (AUROC = 0.784), GCLIPL (AUROC = 0.770), IRL (AUROC = 0.755), and the complete retina (AUROC = 0.752). In contrast, ORL and OPLONL do not appear to be helpful for discriminating POAG. Conclusions: Some results of PP software may be useful for discriminating POAG

Glaucomatous Maculopathy: Thickness Differences on Inner and Outer Macular Layers between Ocular Hypertension and Early Primary Open-Angle Glaucoma Using 8 × 8 Posterior Pole Algorithm of SD-OCT

The purpose of this study was to compare the thickness of all inner and outer macular layers between ocular hypertension (OHT) and early primary open-angle glaucoma (POAG) using spectral domain optical coherence tomography (SD-OCT) 8 &times; 8 posterior pole algorithm (8 &times; 8 PPA). Fifty-seven eyes of 57 OHT individuals and fifty-seven eyes of 57 early POAG patients were included. The thickness of macular retinal nerve fiber layer (mRNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform and nuclear layer, photoreceptor layer (PRL) and retinal pigment epithelium were obtained in 64 cells for each macular layer and mean thickness of superior and inferior hemispheres was also calculated. Thinning of superior and inferior hemisphere mean thickness in mRNFL, GCL and IPL and thickening of superior and inferior hemisphere mean thickness in PRL and inferior hemisphere in INL were found in early GPAA group. Otherwise, heatmaps representing cell-to-cell comparisons showed thinning patterns in inner retinal layers (except for INL) and thickening patterns in outer retinal layers in GPAA group. We found that 8 &times; 8 PPA not only allows the detection of significant thinning patterns in inner retinal layers, but also thickening patterns in outer retinal layers when comparing early POAG eyes to OHT eyes

RBR-type E3 ligases and the ubiquitin-conjugating enzyme UBC26 regulate abscisic acid receptor levels and signaling

The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING finger ABA-related 1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)-type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified Cys361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-Receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared to the wild type. Together our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin (Ub) at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases. Abscisic acid receptors are targeted for degradation by a family of E3 ubiquitin ligases at different subcellular locations, which modulates hormone signaling in plasma membrane, cytosol, and nucleu

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

<div><p>Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα^−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p> </div