Clinical-grade peptide-based inhibition of CK2 blocks viability and proliferation of T-ALL cells and counteracts IL-7 stimulation and stromal support

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Despite remarkable advances in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), relapsed cases are still a major challenge. Moreover, even successful cases often face long-term treatment-associated toxicities. Targeted therapeutics may overcome these limitations. We have previously demonstrated that casein kinase 2 (CK2)-mediated phosphatase and tensin homologue (PTEN) posttranslational inactivation, and consequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling hyperactivation, leads to increased T-ALL cell survival and proliferation. We also revealed the existence of a crosstalk between CK2 activity and the signaling mediated by interleukin 7 (IL-7), a critical leukemia-supportive cytokine. Here, we evaluated the impact of CIGB-300, a the clinical-grade peptide-based CK2 inhibitor CIGB-300 on T-ALL biology. We demonstrate that CIGB-300 decreases the viability and proliferation of T-ALL cell lines and diagnostic patient samples. Moreover, CIGB-300 overcomes IL-7-mediated T-ALL cell growth and viability, while preventing the positive effects of OP9-delta-like 1 (DL1) stromal support on leukemia cells. Signaling and pull-down experiments indicate that the CK2 substrate nucleophosmin 1 (B23/NPM1) and CK2 itself are the molecular targets for CIGB-300 in T-ALL cells. However, B23/NPM1 silencing only partially recapitulates the anti-leukemia effects of the peptide, suggesting that CIGB-300-mediated direct binding to CK2, and consequent CK2 inactivation, is the mechanism by which CIGB-300 downregulates PTEN S380 phosphorylation and inhibits PI3K/Akt signaling pathway. In the context of IL-7 stimulation, CIGB-300 blocks janus kinase / signal transducer and activator of transcription (JAK/STAT) signaling pathway in T-ALL cells. Altogether, our results strengthen the case for anti-CK2 therapeutic intervention in T-ALL, demonstrating that CIGB-300 (given its ability to circumvent the effects of pro-leukemic microenvironmental cues) may be a valid tool for clinical intervention in this aggressive malignancy.This work was supported by the consolidator grant ERC CoG-648455 from the European Research Council, under the European Union’s Horizon 2020 research and innovation program, and FAPESP/20015/2014 and PTDC/MEC-HEM/31588/2017 grants from Fundação para a Ciência e a Tecnologia (FCT), to JTB.info:eu-repo/semantics/publishedVersio

Características morfológicas e histológicas de los ovarios de dos grupos genéticos de cuyes (Cavia porcellus) de América del Sur

This study aimed to describe morphological and histological characteristics of ovaries of two genetic groups of guinea pigs from Latin America. Ovaries from 20 improved and 20 native guinea pigs were collected after slaughtering. One ovary from each animal was weighed, measured, counted for visible follicles on the ovarian surface, and used for subsequent oocyte collection by the slicing method. Contralateral ovaries were used to prepare histological sections and quantify follicles. Body and ovary weight and ovary length were significantly greater in improved than in native guinea pigs (p<0.01). Ovarian weight was greater in diestrus than in proestrus (p=0.0632) only in improved animals. The number of primordial, primary, secondary, and total follicles did not differ between genetic groups. The number of antral follicles was significantly greater in improved than in native guinea pigs. The thickness of zona pellucida and oocyte diameter did not differ between groups. The thickness of the zona pellucida was significantly greater in oocytes of category A than B and C in both groups of guinea pigs. In conclusion, ovaries from improved guinea pigs were heavier and longer than those from native animals. The number of antral follicles was greater in improved than native guinea pigs. Zona pellucida thickness and oocyte diameter were similar in both groups of guinea pigs.El estudio tuvo como objetivo describir las características morfológicas e histológicas de los ovarios de dos grupos genéticos de cuyes de América Latina. Se recolectaron ovarios de 20 cuyes mejorados y 20 nativos después del sacrificio. Se pesó y midió un ovario de cada animal, se contaron los folículos visibles en la superficie ovárica y se colectaron los ovocitos mediante cortes seriados de la corteza ovárica. Los ovarios contralaterales se utilizaron para preparar cortes histológicos y cuantificar los folículos. El peso corporal y la longitud y peso de los ovarios fueron significativamente mayores en los cuyes mejorados que en los nativos (p<0.01). El peso de los ovarios fue mayor en diestro que en proestro (p=0.0632) en los animales mejorados. El número de folículos primordiales, primarios, secundarios y totales no difirió entre los grupos genéticos. El número de folículos antrales fue significativamente mayor en los cuyes mejorados que en los nativos. El grosor de la zona pelúcida y el diámetro de los ovocitos no difirieron entre los grupos. El grosor de la zona pelúcida fue significativamente mayor en los ovocitos de la categoría A que en los de la B y C en ambos grupos de cuyes. En conclusión, los ovarios de los cuyes mejorados fueron más pesados y largos que los de los animales nativos. El número de folículos antrales fue mayor en los cutes mejorados que en los nativos. El grosor de la zona pelúcida y el diámetro de los ovocitos fueron similares en ambos grupos de cuyes

Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization

CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization.Fil: Perera, Yasser. Centro de Ingeniería Genética y Biotecnología; CubaFil: Costales, Heydi C.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Diaz, Yakelin. Centro de Ingeniería Genética y Biotecnología; CubaFil: Reyes, Osvaldo. Centro de Ingeniería Genética y Biotecnología; CubaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mendez, Lissandra. Centro de Ingeniería Genética y Biotecnología; CubaFil: Gómez, Roberto E.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Acevedo, Boris E.. Centro de Ingeniería Genética y Biotecnología; CubaFil: Gomez, Daniel Eduardo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perea, Silvio E.. Centro de Ingeniería Genética y Biotecnología; Cub

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.Fil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Benavent Acero, Fernando Rodrigo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Perera, Yasser. Center for Genetic Engineering and Biotechnology; CubaFil: Rodríguez, Arielis. Center for Genetic Engineering and Biotechnology; CubaFil: Perea, Silvio E.. Center for Genetic Engineering and Biotechnology; CubaFil: Acevedo Castro, Boris. Center for Genetic Engineering and Biotechnology; CubaFil: Gomez, Roberto. No especifíca;Fil: Alonso, Daniel F.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Gomez, Daniel Eduardo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The Effect of Prior Exposures on the Notched Fatigue Behavior of Disk Superalloy ME3

Environmental attack has the potential to limit turbine disk durability, particularly in next generation engines which will run hotter; there is a need to understand better oxidation at potential service conditions and develop models that link microstructure to fatigue response. More efficient gas turbine engine designs will require higher operating temperatures. Turbine disks are regarded as critical flight safety components; a failure is a serious hazard. Low cycle fatigue is an important design criteria for turbine disks. Powder metallurgy alloys, like ME3, have led to major improvements in temperature performance through refractory additions (e.g. Mo,W) at the expense of environmental resistance (Al, Cr). Service conditions for aerospace disks can produce major cycle periods extending from minutes to hours and days with total service times exceeding 1,000 hours in aerospace applications. Some of the effects of service can be captured by extended exposures at elevated temperature prior to LCF testing. Some details of the work presented here have been published

Preclinical efficacy of CIGB-300, an anti-CK2 peptide, on breast cancer metastasic colonization

CK2 is a serine/threonine kinase that is overexpressed in breast cancer and its inhibition is associated to reduced tumor growth and disease progression. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to protein kinase CK2 catalytic subunit alpha and to CK2 substrates thus preventing the enzyme activity. Our aim was to evaluate the potential therapeutic benefits of CIGB-300 on breast cancer disease using experimental models with translational relevance. We demonstrated that CIGB-300 reduces breast cancer cell growth in MDA-MB-231, MCF-7 and F3II cells, exerting a pro-apoptotic action and cell cycle arrest. We also found that CIGB-300 decreased cell adhesion, migration and clonogenic capacity of malignant cells. Effect on experimental breast cancer lung metastasis was evaluated after surgical removal of primary F3II tumors or after tail vein injection of tumor cells, also we evaluated CIGB-300 effect on spontaneous lung metastasis in an orthotopic model. Systemic CIGB-300 treatment inhibited breast cancer colonization of the lung, reducing the size and number of metastatic lesions. The present preclinical study establishes for the first time the efficacy of CIGB-300 on breast cancer. These encouraging results suggest that CIGB-300 could be used for the management of breast cancer as an adjuvant therapy after surgery, limiting tumor metastatic spread and thus protecting the patient from distant recurrence.Fil: Gottardo, María Florencia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Capobianco, Carla Sabrina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sidabra, Johanna Elena. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garona, Juan. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perera, Yasser. Centro de Ingeniería Genética y Biotecnología ; CubaFil: Perea, Silvio E.. Centro de Ingeniería Genética y Biotecnología ; CubaFil: Alonso, Daniel Fernando. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Farina, Hernán Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Experimental conditions for microhabitat selection in terrestrial snails

Habitat selection by terrestrial snails, used as laboratory animals, is an important factor because it can alter growth rates often used as a good biological indicator, moreover of its productive meaning. As support to the studies in this field, different models from election of microhabitat based on the individual selection or derived from other questions as the availability or the accessibility of the habitat or patron of movement of the organism are reviewed. Finally two experimental designs used in invertebrates under laboratorial conditions for preference studies are considered.La selección de hábitat por los caracoles terrestres que se utilizan como animales de experimentación, es importante porque condiciona el crecimiento y éste, además de su importancia productiva, es un buen indicador en muchos estudios. Como apoyo a los estudios en este campo, se revisan diferentes modelos de elección de microhábitat en función de la selección del propio individuo o bien derivados de cuestiones en buena parte ajenas a él como la disponibilidad o accesibilidad del hábitat o el patrón de movimiento del organismo. Finalmente se plantean dos diseños experimentales adecuados para el estudio de preferencia en invertebrados bajo condiciones de laboratorio

Efecto de la adición de melatonina en el medio de maduración y/o vitrificación de ovocitos sobre la producción in vitro de embriones bovinos

The effect of the addition of melatonin (Mt) on the maturation and / or vitrification of bovine oocytes on cleavage and subsequent embryonic development was evaluated. Oocyte-cumulus cell complexes (COCs) were obtained from creole cows by ovum pick up (OPU) and by follicular aspiration from abattoir ovaries (AO). From the pool of oocytes obtained from both sources, those with homogenous cytoplasm and three or more compact layers of cluster cells were selected. The selected COCs were randomly assigned to five treatments: T1, matured with Mt and vitrified without Mt; T2, matured and vitrified with Mt; T3, matured without Mt and vitrified with Mt; T4 (control) matured and vitrified without Mt; T5, matured without Mt and not vitrified. The concentration of Mt in the maturation and vitrification media was 0.01 μM (10-9 M). The oocytes were matured, vitrified, fertilized and the presumed zygotes cultured until day 7 post-fertilization in vitro. The data were analysed by logistic regression. Regardless of the origin of the oocytes, cleavage rate (C) and embryo production (EP) was similar between treatments. The C in the OPU oocytes was greater in T4, and the EP was similar between treatments. In AO, the results did not vary between treatment in C and EP. In conclusion, Mt reduced the C in oocytes collected by OPU, while it did not affect EP. In the AO oocyte source, the addition of Mt did not affect the C nor the EP.Se evaluó el efecto de la adición de melatonina (Mt) en el medio de maduración y/o de vitrificación de ovocitos bovinos sobre clivaje y posterior desarrollo embrionario. Los complejos ovocito-células del cúmulo (COCs) fueron obtenidos de vacas criollas mediante la técnica de aspiración transvaginal guiada por ultrasonografía (OPU) y de ovarios de matadero (OM). Del pool de ovocitos obtenidos de ambas fuentes se seleccionaron los que tenían citoplasma homogéneo y tres o más capas compactas de células del cúmulo. Los COCs seleccionados fueron asignados aleatoriamente a cinco tratamientos: T1, madurados con Mt y vitrificados sin Mt; T2, madurados y vitrificados con Mt; T3, madurados sin Mt y vitrificados con Mt; T4 (control) madurados y vitrificados sin Mt; T5, madurados sin Mt y no vitrificados. La concentración de Mt en los medios de maduración y vitrificación fue de 0.01 μM (10-9 M). Los ovocitos fueron madurados, vitrificados, fecundados y los presuntos cigotos cultivados hasta el día 7 pos-fecundación in vitro. Los datos fueron analizados por regresión logística. Independientemente del origen de los ovocitos, el porcentaje de clivaje (PC) y de producción de embriones (PIV) fue similar entre tratamientos. El PC en los ovocitos de OPU fue mayor en T4, y la PIV fue similar entre tratamientos. En los de OM, los resultados no variaron entre tratamiento en PC y PIV. En conclusión, la Mt redujo el PC en ovocitos colectados por OPU, mientras que no afectó la PIV. En los colectados de OM la adición de Mt no afectó el PC ni la PI

Nanometer-Scale Chemistry of a Calcite Biomineralization Template: Implications for Skeletal Composition and Nucleation

Plankton, corals, and other organisms produce calcium carbonate skeletons that are integral to their survival, form a key component of the global carbon cycle, and record an archive of past oceanographic conditions in their geochemistry. A key aspect of the formation of these biominerals is the interaction between organic templating structures and mineral precipitation processes. Laboratory-based studies have shown that these atomic-scale processes can profoundly influence the architecture and composition of minerals, but their importance in calcifying organisms is poorly understood because it is difficult to measure the chemistry of in vivo biomineral interfaces at spatially relevant scales. Understanding the role of templates in biomineral nucleation, and their importance in skeletal geochemistry requires an integrated, multiscale approach, which can place atom-scale observations of organic-mineral interfaces within a broader structural and geochemical context. Here we map the chemistry of an embedded organic template structure within a carbonate skeleton of the foraminifera Orbulina universa using both atom probe tomography (APT), a 3D chemical imaging technique with Angström-level spatial resolution, and time-of-flight secondary ionization mass spectrometry (ToF-SIMS), a 2D chemical imaging technique with submicron resolution. We quantitatively link these observations, revealing that the organic template in O. universa is uniquely enriched in both Na andMg, and contributes to intraskeletal chemical heterogeneity. Our APT analyses reveal the cation composition of the organic surface, offering evidence to suggest that cations other than Ca2+, previously considered passive spectator ions in biomineral templating, may be important in defining the energetics of carbonate nucleation on organic template

Silicon-lattice-matched boron-doped gallium phosphide: A scalable acousto-optic platform

The compact size, scalability, and strongly confined fields in integrated photonic devices enable new functionalities in photonic networking and information processing, both classical and quantum. Gallium phosphide (GaP) is a promising material for active integrated photonics due to its high refractive index, wide band gap, strong nonlinear properties, and large acousto-optic figure of merit. In this work we demonstrate that silicon-lattice-matched boron-doped GaP (BGaP), grown at the 12-inch wafer scale, provides similar functionalities as GaP. BGaP optical resonators exhibit intrinsic quality factors exceeding 25,000 and 200,000 at visible and telecom wavelengths respectively. We further demonstrate the electromechanical generation of low-loss acoustic waves and an integrated acousto-optic (AO) modulator. High-resolution spatial and compositional mapping, combined with ab initio calculations indicate two candidates for the excess optical loss in the visible band: the silicon-GaP interface and boron dimers. These results demonstrate the promise of the BGaP material platform for the development of scalable AO technologies at telecom and provide potential pathways toward higher performance at shorter wavelengths