Genetic variant panel allows predicting both obesity risk, and efficacy of procedures and diet in weight loss

PurposeObesity is a multifactorial condition with a relevant genetic correlation. Recent advances in genomic research have identified several single nucleotide polymorphisms (SNPs) in genes such as FTO, MCM6, HLA, and MC4R, associated with obesity. This study aimed to evaluate the association of 102 SNPs with BMI and weight loss treatment response in a multi-ethnic population.MethodsThe study analyzed 9,372 patients for the correlation between SNPs and BMI (dataset A). The correlation between SNP and weight loss was accessed in 474 patients undergoing different treatments (dataset B). Patients in dataset B were further divided into 3 categories based on the type of intervention: dietary therapy, intragastric balloon procedures, or surgeries. SNP association analysis and multiple models of inheritance were performed.ResultsIn dataset A, ten SNPs, including rs9939609 (FTO), rs4988235 (MCM6), and rs2395182 (HLA), were significantly associated with increased BMI. Additionally, other four SNPs, rs7903146 (TCF7L2), (rs6511720), rs5400 (SLC2A2), and rs7498665 (SH2B1), showed sex-specific correlation. For dataset B, SNPs rs2016520 (PPAR-Delta) and rs2419621 (ACSL5) demonstrated significant correlation with weight loss for all treatment types. In patients who adhered to dietary therapy, SNPs rs6544713 (ABCG8) and rs762551 (CYP1A2) were strongly correlated with weight loss. Patients undergoing surgical or endoscopic procedures exhibited differential correlations with several SNPs, including rs1801725 (CASR) and rs12970134 (MC4R), and weight loss.ConclusionThis study provides valuable insights into the genetic factors influencing BMI and weight loss response to different treatments. The findings highlight the potential for personalized weight management approaches based on individual genetic profiles

Hypoxia Reduces Cell Attachment of SARS-CoV-2 Spike Protein by Modulating the Expression of ACE2, Neuropilin-1, Syndecan-1 and Cellular Heparan Sulfate

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1αdependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), the European Research Council (ERC) (grant numbers: ERC-2018-StG 804236-NEXTGEN-IO to A.P and ERC-2017-AdG 788143-RECGLYCANMR to J.J-B.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the FERO Foundation. Personal fellowships: E.P. (Juan de la Cierva-Formación, FJC2018-035449-I), L.V. (Juan de la Cierva-Formación, FJCI-2017-34099), A.B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), A.G. (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), A.A (La Caixa Inphinit, LCF/BQ/DR20/11790022), B.J. (Basque Government, PRE_2019_1_0320), L.M. (Juan de la Cierva-Formación, FJC2019-039983-I), E.B. (MINECO, BFU2016-76872-R; Excellence Networks, SAF2017-90794-REDT) and A.P. (Ramón y Cajal, RYC2018-024183-I; Proyectos I+D+I, PID2019-107956RA-I00; and Ikerbasque Research Associate)

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

Desarrollo de un modelo in vitro de barrera intestinal para la evaluación del riesgo de los nanomateriales

El campo de la nanotecnología está expandiéndose de forma exponencial y con ello la preocupación social sobre los posibles efectos de sus productos (nanomateriales; NMs) en los organismos vivos. Dado que las principales vías de exposición a los NMs son el contacto dermal, la inhalación y la ingestión, la barrera intestinal adquiere gran importancia debido a la presencia de NMs en comida y envases de comida, además de su presencia en muchos otros productos de uso diario. En este contexto, el objetivo de esta Tesis es desarrollar un modelo intestinal formado por células Caco-2 para la evaluación del riesgo de NMs. Las células Caco-2 son células de adenocarcinoma de colon que tienen la capacidad de diferenciarse en enterocitos del intestino delgado. Bajo el marco del proyecto NANoREG, los resultados obtenidos han demostrado que se ha desarrollado con éxito un protocolo robusto para obtener una barrera in vitro intestinal estable y reproducible compuesta por estas células Caco-2 diferenciadas. Usando concentraciones subtóxicas de TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPs y MWCNT durante 24 horas, se han realizado diferentes tipos de análisis, a saber: a) evaluación de la integridad y permeabilidad de la monocapa, b) internalización celular, c) translocación de los NMs, d) evaluación del daño genotóxico y d) evaluación de la integridad de la monocapa a través de cambios de la expresión del mRNA. Los resultados tras la exposición a concentraciones subtóxicas no mostraron alteración en la integridad ni en la permeabilidad de la monocapa. Además, se observó internalización de las AgNPs tanto en el citoplasma como en el núcleo, mientras que las TiO2NPs y las CeO2NPs se encontraron sedimentadas en la membrana apical de las células. La capacidad de evaluar la translocación de los NMs a través de la barrera celular resultó ser muy limitada. El TEM-EDX, la microscopía confocal y el ICP-MS produjeron resultados de los que se puede concluir una limitada translocación de TiO2NPs, ZnONPS y CeO2NPS. En la evaluación del daño genotóxico mediante el ensayo del cometa, se observó que sólo las AgNPs son capaces de producir daño oxidativo en el DNA y tan sólo en la concentración más alta analizada (50 μg/mL). En la evaluación de la expresión del mRNA de genes de transportadores celulares (SI y SLC15A1) y de adhesión célula-célula (OCCLUDIN y CLAUDIN2), los resultados mostraron mucha variabilidad entre réplicas y no se encontraron cambios significativos en ninguno de ellos tras la exposición a AgNPs. Al margen del proyecto europeo y con el fin de evaluar el riesgo de la exposición a NMs a largo plazo, se expusieron las células Caco-2 en su estado no diferenciado a concentraciones subtóxicas de AgNPs durante 6 semanas evaluándose su capacidad de transformación. Estas células mostraron necesitar un tiempo de adaptación en el que disminuyen el ritmo de división debido a la exposición hasta que lo recuperan a lo largo del tratamiento. Tras 6 semanas de exposición, las células Caco-2 expresan algunas características de célula transformada: a) aumentan su nivel de proliferación, b) adquieren la capacidad de crecer en soft-agar, y de promover el crecimiento en soft-agar de otras células tumorales (HCT116), c) aumentan la secreción de MMPs, y d) incrementan su capacidad migratoria. Sin embargo, la exposición no mostró inducir cambios en la expresión de los genes involucrados en EMT, adhesión celular, citoesqueleto o supresores de tumores. A pesar de esto, los resultados globales indicarían que las AgNPs no son seguras en términos de carcinogénesis.The field of nanotechnology is increasing every day. For this reason, the social concern about the possible effects of their products (nanomaterials; NMs) in living organisms is also growing. Since the main routes of exposure to NMs are dermal contact, inhalation, and ingestion, the intestinal barrier is very important due to the presence of NMs in food and food packaging, in addition to its presence in many other daily products. In this context, the objective of this Thesis is to develop an intestinal model formed by Caco-2 cells in order to assess the risk of NMs. Caco-2 cells are from colon adenocarcinoma and have the capacity to differentiate into enterocytes of the small intestine. Under the framework of the NANoREG’s project, the results have shown that we have successfully developed a robust protocol to obtain stable and reproducible in vitro intestinal barrier composed of these differentiated Caco-2 cells. Using sub-toxic concentrations of TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPS and MWCNT for 24 hours, we have performed different types of analysis: a) assessment of the integrity and permeability of the monolayer, b) cellular internalization, c) translocation of NMs, d) evaluation of genotoxic damage, d) assessment of the integrity of the monolayer through changes in mRNA expression. The results after exposure to sub-toxic concentrations showed no alteration in the integrity or the permeability of the monolayer. Furthermore, internalization of AgNPS was observed in both the cytoplasm and nucleus while TiO2NPs and CeO2NPs found sedimented on the apical membrane of cells. The ability to evaluate the translocation of NMs through the cell barrier proved to be very limited. TEM-EDX, confocal microscopy, and ICP-MS showed limited translocation of TiO2NPs, ZnONPs, and CeO2NPS. In the evaluation of genotoxic damage by comet assay, it was observed that only AgNPS are capable of producing oxidative DNA damage and only at the highest concentration tested (50 μg/mL). In assessing mRNA expression of genes of cellular transporters (SI and SLC15A1) and cell-cell adhesion (OCCLUDIN and CLAUDIN2), results showed a lot of variability between replicates and no significant changes were found in any of them after exposure to AgNPS. Besides the European project and in order to assess the risk of exposure to NMs in the long term, Caco-2 cells in their undifferentiated state were exposed for 6 weeks to sub-toxic concentrations of AgNPS evaluating its transformation capacity. These cells were subjected to a period of adaptation in which the cells decreased the rate of division due to exposure until regaining it at the end of the treatment. After 6 weeks of exposure, the Caco-2 cells expressed some features of the transformed cells: a) increased the proliferation rate, b) acquired the ability to grow in soft agar, and promoted the growth of other tumour cells (HCT116) in soft-agar, c) increased the secretion of MMPs, and d) increased their migratory capacity. However, exposure didn’t show changes in the expression of genes involved in EMT, cell adhesion, cytoskeleton or tumour suppressors. Despite this, the overall results indicate that the AgNPS are not safe in terms of carcinogenesis

Desarrollo de un modelo in vitro de barrera intestinal para la evaluación del riesgo de los nanomateriales

El campo de la nanotecnología está expandiéndose de forma exponencial y con ello la preocupación social sobre los posibles efectos de sus productos (nanomateriales; NMs) en los organismos vivos. Dado que las principales vías de exposición a los NMs son el contacto dermal, la inhalación y la ingestión, la barrera intestinal adquiere gran importancia debido a la presencia de NMs en comida y envases de comida, además de su presencia en muchos otros productos de uso diario. En este contexto, el objetivo de esta Tesis es desarrollar un modelo intestinal formado por células Caco-2 para la evaluación del riesgo de NMs. Las células Caco-2 son células de adenocarcinoma de colon que tienen la capacidad de diferenciarse en enterocitos del intestino delgado. Bajo el marco del proyecto NANoREG, los resultados obtenidos han demostrado que se ha desarrollado con éxito un protocolo robusto para obtener una barrera in vitro intestinal estable y reproducible compuesta por estas células Caco-2 diferenciadas. Usando concentraciones subtóxicas de TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPs y MWCNT durante 24 horas, se han realizado diferentes tipos de análisis, a saber: a) evaluación de la integridad y permeabilidad de la monocapa, b) internalización celular, c) translocación de los NMs, d) evaluación del daño genotóxico y d) evaluación de la integridad de la monocapa a través de cambios de la expresión del mRNA. Los resultados tras la exposición a concentraciones subtóxicas no mostraron alteración en la integridad ni en la permeabilidad de la monocapa. Además, se observó internalización de las AgNPs tanto en el citoplasma como en el núcleo, mientras que las TiO2NPs y las CeO2NPs se encontraron sedimentadas en la membrana apical de las células. La capacidad de evaluar la translocación de los NMs a través de la barrera celular resultó ser muy limitada. El TEM-EDX, la microscopía confocal y el ICP-MS produjeron resultados de los que se puede concluir una limitada translocación de TiO2NPs, ZnONPS y CeO2NPS. En la evaluación del daño genotóxico mediante el ensayo del cometa, se observó que sólo las AgNPs son capaces de producir daño oxidativo en el DNA y tan sólo en la concentración más alta analizada (50 μg/mL). En la evaluación de la expresión del mRNA de genes de transportadores celulares (SI y SLC15A1) y de adhesión célula-célula (OCCLUDIN y CLAUDIN2), los resultados mostraron mucha variabilidad entre réplicas y no se encontraron cambios significativos en ninguno de ellos tras la exposición a AgNPs. Al margen del proyecto europeo y con el fin de evaluar el riesgo de la exposición a NMs a largo plazo, se expusieron las células Caco-2 en su estado no diferenciado a concentraciones subtóxicas de AgNPs durante 6 semanas evaluándose su capacidad de transformación. Estas células mostraron necesitar un tiempo de adaptación en el que disminuyen el ritmo de división debido a la exposición hasta que lo recuperan a lo largo del tratamiento. Tras 6 semanas de exposición, las células Caco-2 expresan algunas características de célula transformada: a) aumentan su nivel de proliferación, b) adquieren la capacidad de crecer en soft-agar, y de promover el crecimiento en soft-agar de otras células tumorales (HCT116), c) aumentan la secreción de 12Ps, y d) incrementan su capacidad migratoria. Sin embargo, la exposición no mostró inducir cambios en la expresión de los genes involucrados en EMT, adhesión celular, citoesqueleto o supresores de tumores. A pesar de esto, los resultados globales indicarían que las AgNPs no son seguras en términos de carcinogénesis.The field of nanotechnology is increasing every day. For this reason, the social concern about the possible effects of their products (nanomaterials; NMs) in living organisms is also growing. Since the main routes of exposure to NMs are dermal contact, inhalation, and ingestion, the intestinal barrier is very important due to the presence of NMs in food and food packaging, in addition to its presence in many other daily products. In this context, the objective of this Thesis is to develop an intestinal model formed by Caco-2 cells in order to assess the risk of NMs. Caco-2 cells are from colon adenocarcinoma and have the capacity to differentiate into enterocytes of the small intestine. Under the framework of the NANoREG's project, the results have shown that we have successfully developed a robust protocol to obtain stable and reproducible in vitro intestinal barrier composed of these differentiated Caco-2 cells. Using sub-toxic concentrations of TiO2NPs, ZnONPs, SiO2NPs, CeO2NPs, AgNPS and MWCNT for 24 hours, we have performed different types of analysis: a) assessment of the integrity and permeability of the monolayer, b) cellular internalization, c) translocation of NMs, d) evaluation of genotoxic damage, d) assessment of the integrity of the monolayer through changes in mRNA expression. The results after exposure to sub-toxic concentrations showed no alteration in the integrity or the permeability of the monolayer. Furthermore, internalization of AgNPS was observed in both the cytoplasm and nucleus while TiO2NPs and CeO2NPs found sedimented on the apical membrane of cells. The ability to evaluate the translocation of NMs through the cell barrier proved to be very limited. TEM-EDX, confocal microscopy, and ICP-MS showed limited translocation of TiO2NPs, ZnONPs, and CeO2NPS. In the evaluation of genotoxic damage by comet assay, it was observed that only AgNPS are capable of producing oxidative DNA damage and only at the highest concentration tested (50 μg/mL). In assessing mRNA expression of genes of cellular transporters (SI and SLC15A1) and cell-cell adhesion (OCCLUDIN and CLAUDIN2), results showed a lot of variability between replicates and no significant changes were found in any of them after exposure to AgNPS. Besides the European project and in order to assess the risk of exposure to NMs in the long term, Caco-2 cells in their undifferentiated state were exposed for 6 weeks to sub-toxic concentrations of AgNPS evaluating its transformation capacity. These cells were subjected to a period of adaptation in which the cells decreased the rate of division due to exposure until regaining it at the end of the treatment. After 6 weeks of exposure, the Caco-2 cells expressed some features of the transformed cells: a) increased the proliferation rate, b) acquired the ability to grow in soft agar, and promoted the growth of other tumour cells (HCT116) in soft-agar, c) increased the secretion of MMPs, and d) increased their migratory capacity. However, exposure didn't show changes in the expression of genes involved in EMT, cell adhesion, cytoskeleton or tumour suppressors. Despite this, the overall results indicate that the AgNPS are not safe in terms of carcinogenesis