VERIFICACIÓN DEL MÉTODO ANALÍTICO DE ESPECTROSCOPÍA DE ABSORCIÓN ATÓMICA CON HORNO DE GRAFITO PARA LA CUANTIFICACIÓN DE CADMIO EN ALMENDRA DE CACAO (Theobroma cacao)

El método de espectroscopía de absorción atómica (AA) de llama para la determinación de cadmio (Cd) en almendra de cacao (Theobroma cacao) utilizado por Agrocalidad es tóxico para el ser humano y el ambiente; por ello, se pretende utilizar el método de espectroscopia de absorción atómica con horno de grafito (GFAAS) por ser más confiable y seguro. Así, se realizó la verificación de cuatro parámetros de desempeño del método GFAAS para cuantificar Cd en almendra de cacao utilizando material de referencia certificado (MRC) y muestras provenientes de cuatro fincas (A, B, C, D) ubicadas en la zona cacaotera de Ecuador, cantón Flavio Alfaro, provincia de Manabí. Se realizó una prueba inter-laboratorios y finalmente se elaboró el protocolo (PEE/B/14). Sobre el MRC (Cód. 07206B y 07167A) se verificó: linealidad, precisión, veracidad e incertidumbre de acuerdo con la Guía Eurachem de Eurolab España y Morillas (2016), y con el estándar IRAM 35050 (2001) se encontró linealidad entre 0 y 8 ppb con R2 = 0;9988; desviación estándar de 0,0005 y 0,0022 respectivamente; sesgo en 0,007 y porcentaje de recuperación de 109,75; la incertidumbre estándar de 0,00013 y 0,00082. El contenido de Cd en las muestras de la finca A con 0,54 ppm, las Fincas B-D con 0,26 ppm y 0,15 ppm en la finca C. En la prueba inter-laboratorios se estableció la misma concentración de cadmio para la muestra C3 y, de acuerdo con lo estipulado por la Unión Europea, el cacao de las cuatro fincas podría ser exportado sin restricciones.//The flame atomic absorption (AA) spectroscopy method for the determination of cadmium (Cd) in cocoa almond (Theobroma cacao) used by Agrocalidad is toxic to humans and the environment, reason for which the atomic absorption spectroscopy method with graphite furnace (GFAAS) was used, because it is more reliable and safer. Thus, four performance parameters of GFAAS method were used to quantify Cd in cocoa almond, by using certified reference material (MRC) and samples from four farms (A, B, C, D) located in the most important cocoa area of Ecuador, Flavio Alfaro city, province of Manabi. An interlaboratory test was performed and finally a protocol (PEE/B/14) was developed. Using the MRC (Code 07806B and 07167) was verified: linearity, precision, veracity and uncertainty in accordance with international standards, Eurachem Guide of Eurolab Spain and Morillas (2016), and with the standard IRAM 35050 (2001) were found linearity between 0 and 8 ppb with R2 = 0;9988; standard deviation 0.0005 and 0.0022, respectively; slant was 0.007 and the recovery percentage was 109.75; standard uncertainty was 0.00013 and 0.00082. The content of Cd in samples from farm A was 0.54 ppm, B and D farms 0.26 ppm and 0.15 ppm in farm C. In the interlaboratory test, the same concentration of Cd was established for simple C3 and, in accordance with the stipulated by the European Union, cocoa from the four farms could be exported without restrictions

New insights into cancer: MDM2 binds to the citrullinating enzyme PADI4

PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. MDM2 is an E3 ubiquitin ligase which is crucial for down-regulation of degradation of the tumor suppressor gene p53. Given the relationship between both PADI4 and MDM2 with p53-signaling pathways, we hypothesized they may interact directly, and this interaction could be relevant in the context of cancer. Here, we showed their association in the nucleus and cytosol in several cancer cell lines. Furthermore, binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that MDM2 could bind to the active site of PADI4, as confirmed by in silico experiments. In vitro and in silico studies showed that the isolated N-terminal region of MDM2, N-MDM2, interacted with PADI4, and residues Thr26, Val28, Phe91 and Lys98 were more affected by the presence of the enzyme. Moreover, the dissociation constant between N-MDM2 and PADI4 was comparable to the IC50 of GSK484 from in cellulo experiments. The interaction between MDM2 and PADI4 might imply MDM2 citrullination, with potential therapeutic relevance for improving cancer treatment, due to the generation of new antigens

The intrinsically disordered, epigenetic factor RYBP binds to the citrullinating enzyme PADI4 in cancer cells

14 p.-6 fig.-1 tab.RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 μM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.This research was funded by Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033/ and “ERDF A way of Making Europe” [PID2021-127296OB-I00 to AVC; and PDC2022-133952-I00 to EF]; by Instituto de Salud Carlos III co-funded by European Social Fund “Investing in your future” [CP19/00095 to CdJ] [PI22/00824 to MS and CdJ] [PI18/00394 to OA]; by Diputación General de Aragón [“Protein targets and Bioactive Compounds group” E45-20R to AVC, and “Digestive Pathology Group” B25-20R to OA], and by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana) [CAICO 2021/0135 to CdJ and JLN].Peer reviewe

Verificación del método analítico de espectroscopia de absorción atómica con horno de grafito para la cuantificación de cadmio en almendra de cacao (Theobroma cacao).

The objective of this work was to verify the performance parameters of the analytical method of atomic absorption spectroscopy with graphite furnace for quantification of cadmium in cocoa (Theobroma cacao) almond, for which a protocol was prepared, analyzes were performed on the certified reference material (MRC), the concentration of cadmium in dry cocoa beans four farms (Farm A, B, C and D) located in the cocoa zone of the country, Flavio Alfaro Manabí, was determined and an interlaboratory test was performed. The protocol (PEE / B / 14) is a standard operating procedure for the application of this method in the analysis of cadmium in cocoa almond samples. four parameters on MRC (Code. 07206B and Code. 07167A) were verified: linearity, precision, accuracy and uncertainty according to international standards (Eurachem, 2016 and IRAM 2010); thus, the linear range was set between 0 and 8 ppb, with a coefficient of R2=0.9988 linear regression; the standard deviation was 0.0005 and 0.0022 respectively; the slant was 0.007 and the recovery rate 109.75; the standard uncertainty was 0.00013 and 0.00082; which shows the reliability of the results obtained by the application of this analytical method. The cadmium content in the samples was 0.54 ppm at Farm A, 0.26 ppm Farms B-D and 0.15 ppm in the estate C. In the interlaboratory comparison the same concentration of cadmium for C3 sample was established.El objetivo de este trabajo fue verificar los parámetros de desempeño del método analítico de espectroscopia de absorción atómica con horno de grafito para la cuantificación de cadmio en almendra de cacao (Theobroma cacao), para lo cual se elaboró un protocolo, se realizaron análisis sobre material de referencia certificado (MRC), se determinó la concentración de cadmio en almendras secas de cacao de cuatro fincas (Finca A, B, C y D) ubicadas en la zona cacaotera del país, Flavio Alfaro- Manabí y se realizó una prueba interlaboratorios. El protocolo elaborado (PEE/B/14) es un procedimiento operativo estándar para la aplicación de este método en el análisis de cadmio en muestras de almendra de cacao. Se verificaron cuatro parámetros sobre MRC (Cód. 07206B y Cód. 07167A): linealidad, precisión, veracidad e incertidumbre de acuerdo a estándares internacionales (Eurachem, 2016 e IRAM 2010); así, el intervalo lineal se estableció entre 0 y 8 ppb, con un coeficiente de regresión lineal R2=0,9988; la desviación estándar fue de 0,0005 y 0,0022 respectivamente; el sesgo fue de 0.007 y el porcentaje de recuperación de 109.75; la incertidumbre estándar fue de 0,00013 y 0,00082; lo cual demuestra la confiabilidad de los resultados obtenidos por la aplicación de este método analítico. El contenido de cadmio en las muestras fue de 0.54 ppm en la Finca A, 0.26 ppm en las Fincas B-D y 0.15 ppm en la finca C. En la comparación interlaboratorios se estableció la misma concentración de cadmio para la muestra C3

New therapy for pancreatic cancer based on extracellular vesicles

Pancreatic Ductal Adenocarcinoma (PDAC), is the most common aggressive cancer of the pancreas. The standard care of PDAC includes tumor resection and chemotherapy, but the lack of early diagnosis and the limited response to the treatment worsens the patient’s condition. In order to improve the efficiency of chemotherapy, we look for more efficient systems of drug delivery. We isolated and fully characterized small Extracellular Vesicles (EVs) from the RWP-1 cell line. Our study indicates that the direct incubation method was the most efficient loading protocol and that a minimum total amount of drug triggers an effect on tumor cells. Therefore, we loaded the small EVs with two chemotherapeutic drugs (Temozolomide and EPZ015666) by direct incubation method and the amount of drug loaded was measured by high-performance liquid chromatography (HPLC). Finally, we tested their antiproliferative effect on different cancer cell lines. Moreover, the system is highly dependent on the drug structure and therefore RWP-1 small EVsTMZ were more efficient than RWP-1 small EVsEPZ015666. RWP-1 derived small EVs represent a promising drug delivery tool that can be further investigated in preclinical studies and its combination with PRMT5 inhibitor can be potentially developed in clinical trials for the treatment of PDAC