Ecological determinants of obesity risk in Mexican infants: a scoping review.

BACKGROUND: Childhood obesity is a multifactorial disease. Most of these factors start to develop before birth and worsen throughout life. Therefore, prevention efforts should begin in the first 1000 days of life. This study aimed to quantify published studies on risk factors according to the Six-Cs model of childhood obesity (cell, child, clan, culture, community, and country) and determine which of them have been related to anthropometric indicators of overweight or obesity in children under 2 years of age in Mexico. METHODS: A systematic scoping review (PRISMA-ScR) was performed. PubMed, Scopus, and EBSCOhost databases were reviewed. RESULTS: We found that 88% of the studies were observational. The child and family spheres were the most studied, individually and as a whole. The least studied were community, culture, and country. The main risk factors related to obesity indicators were high birth weight, birth by cesarean section, and inadequate feeding practices, in addition to mothers with obesity and those who underestimate their child's weight, stressful parenting style, and food insecurity in the home, together with living in urban areas, family income, and beliefs about preference for ultra-processed products. CONCLUSION: In Mexico, the study of obesity in early childhood is emerging at the research level. However, further efforts are required to close the knowledge gap at the socioecological level to design evidence-based interventions and reduce early obesity.

INTRODUCCIÓN: La obesidad infantil es una enfermedad multifactorial en la que varios factores comienzan a desarrollarse antes del nacimiento y se agravan a lo largo de la vida. Por ello, los esfuerzos de prevención para evitar su desarrollo deben comenzar durante los primeros 1000 días de vida. Los objetivos de este estudio fueron cuantificar los estudios publicados sobre factores de riesgo según el modelo de obesidad infantil de las 6-Cs (célula, niño, familia, cultura, comunidad y país) y determinar cuáles de ellos se han relacionado con indicadores de sobrepeso u obesidad en niños menores de 2 años en México. MÉTODOS: Se realizó una revisión sistemática de alcance (PRISMA-ScR). Se revisaron las bases de datos de PubMed, Scopus y EBSCOhost. RESULTADOS: Se encontró que el 88% de los estudios fueron de tipo observacional. La esfera niño y familia fueron las más abordadas, tanto individual como en conjunto. Las menos estudiadas fueron comunidad, cultura y país. Los principales factores de riesgo relacionados con indicadores de obesidad fueron alto peso al nacer, nacer por vía cesárea y prácticas inadecuadas de alimentación; además, madres con obesidad y que subestiman el peso del hijo, estilo de crianza presionante e inseguridad alimentaria en el hogar, aunado el vivir en zonas urbanas, ingreso económico-familiar y creencias sobre la preferencia por productos ultraprocesados. CONCLUSIONES: En México, el estudio de obesidad durante los primeros 1000 días es emergente a nivel de investigación, pero se requiere continuar con el esfuerzo para cerrar la brecha de conocimiento a nivel socio-ecológico, diseñar intervenciones basadas en la evidencia y disminuir la obesidad temprana.

Ecological determinants of obesity risk in Mexican infants: a scoping review / Determinantes ecológicos de riesgo de obesidad en lactantes mexicanos: una revisión de alcance

Abstract Background: Childhood obesity is a multifactorial disease. Most of these factors start to develop before birth and worsen throughout life. Therefore, prevention efforts should begin in the first 1000 days of life. This study aimed to quantify published studies on risk factors according to the Six-Cs model of childhood obesity (cell, child, clan, culture, community, and country) and determine which of them have been related to anthropometric indicators of overweight or obesity in children under 2 years of age in Mexico. Methods: A systematic scoping review (PRISMA-ScR) was performed. PubMed, Scopus, and EBSCOhost databases were reviewed. Results: We found that 88% of the studies were observational. The child and family spheres were the most studied, individually and as a whole. The least studied were community, culture, and country. The main risk factors related to obesity indicators were high birth weight, birth by cesarean section, and inadequate feeding practices, in addition to mothers with obesity and those who underestimate their child's weight, stressful parenting style, and food insecurity in the home, together with living in urban areas, family income, and beliefs about preference for ultra-processed products. Conclusion: In Mexico, the study of obesity in early childhood is emerging at the research level. However, further efforts are required to close the knowledge gap at the socioecological level to design evidence-based interventions and reduce early obesity.

Resumen Introducción: La obesidad infantil es una enfermedad multifactorial en la que varios factores comienzan a desarrollarse antes del nacimiento y se agravan a lo largo de la vida. Por ello, los esfuerzos de prevención para evitar su desarrollo deben comenzar durante los primeros 1000 días de vida. Los objetivos de este estudio fueron cuantificar los estudios publicados sobre factores de riesgo según el modelo de obesidad infantil de las 6-Cs (célula, niño, familia, cultura, comunidad y país) y determinar cuáles de ellos se han relacionado con indicadores de sobrepeso u obesidad en niños menores de 2 años en México. Métodos: Se realizó una revisión sistemática de alcance (PRISMA-ScR). Se revisaron las bases de datos de PubMed, Scopus y EBSCOhost. Resultados: Se encontró que el 88% de los estudios fueron de tipo observacional. La esfera niño y familia fueron las más abordadas, tanto individual como en conjunto. Las menos estudiadas fueron comunidad, cultura y país. Los principales factores de riesgo relacionados con indicadores de obesidad fueron alto peso al nacer, nacer por vía cesárea y prácticas inadecuadas de alimentación; además, madres con obesidad y que subestiman el peso del hijo, estilo de crianza presionante e inseguridad alimentaria en el hogar, aunado el vivir en zonas urbanas, ingreso económico-familiar y creencias sobre la preferencia por productos ultraprocesados. Conclusiones: En México, el estudio de obesidad durante los primeros 1000 días es emergente a nivel de investigación, pero se requiere continuar con el esfuerzo para cerrar la brecha de conocimiento a nivel socio-ecológico, diseñar intervenciones basadas en la evidencia y disminuir la obesidad temprana.

Propiedades psicométricas del Children Eating Behavior Questionnaire en niños mexicanos menores de 3 años / Psychometric properties of the Children's Eating Behavior Questionnaire in Mexican children under 3 years of age

Resumen Objetivo: México es uno de los países con mayor prevalencia de obesidad infantil a nivel mundial. Se requieren de instrumentos de valoración integrales válidos para el abordaje del problema. El Children Eating Behavior Questionnaire. (CEBQ) permite evaluar las conductas alimentarias de los niños relacionadas con el riesgo de obesidad, por lo que se planteó el siguiente objetivo evaluar las propiedades psicométricas de la versión original del CEBQ en niños mexicanos de 1 a 3 años. Material y Métodos: 201 madres residentes del noreste de México completaron el CEBQ en centros de salud comunitarios. Se realizó un análisis factorial confirmatorio (AFC) a través de componentes principales y fiabilidad (consistencia interna) con alfa de Cronbach (α). Resultados: Se identificó un modelo con siete factores y 24 ítems, los índices de bondad de ajuste del modelo propuesto fueron adecuados: índice de ajuste comparativo (CFI= 0.90), y error cuadrático medio de aproximación (RMSEA= 0.06). Los factores que integran el CEBQ tienen aceptable confiabilidad internaα > 0.70. Conclusión: El CEBQ en niños mexicanos de 1 a 3 años tiene propiedades psicométricas que lo hace una medida confiable y válida para evaluar comportamientos alimentarios relacionados con el riesgo de obesidad. Se recomienda contrastar este modelo en poblaciones similares.

Abstract Objective: Mexico is one of the countries with the highest prevalence of childhood obesity worldwide. Valid comprehensive assessment instruments are required to address the problem. The Child Eating Behavior Questionnaire(CEBQ) allows the assessment of children's eating behaviors related to obesity risk, so the following objective was to evaluate the psychometric properties of the original version of the CEBQ in Mexican children aged 1 to 3 years. Material and Methods: 201 mothers residing in northeastern Mexico completed the CEBQ in community health centers. A confirmatory factor analysis (CFA) was performed through principal components and reliability (internal consistency) with Cronbach's alpha (α). Results: A model with seven factors and 24 items was identified, the goodness-of-fit indices of the proposed model were adequate: comparative fit index (CFI=0.90), and root mean square error of approximation (RMSEA= 0.06). The factors that make up the CEBQ have acceptable internal reliability α > 0.70. Conclusion: The CEBQ in Mexican children aged 1 to 3 years has psychometric properties that make it a reliable and valid measure to assess eating behaviors related to obesity risk. It is recommended to contrast this model in similar populations.

Aquaporins in Fetal Development.

Water homeostasis is essential for fetal growth, and it depends on the successful development of the placenta. Many aquaporins (AQPs) were identified from blastocyst stages to term placenta. In the last years, cytokines, hormones, second messengers, intracellular pH, and membrane proteins were found to regulate their expression and function in the human placenta and fetal membranes. Accumulated data suggest that these proteins may be involved not only in the maintenance of the amniotic fluid volume homeostasis but also in the development of the placenta and fetal organs. In this sense, dysregulation of placental AQPs is associated with gestational disorders. Thus, current evidence shows that AQPs may collaborate in cellular events including trophoblast migration and apoptosis. In addition, aquaglyceroporins are involved in energy metabolism as well as urea elimination across the placenta. In the last year, the presence of AQP9 in trophoblast mitochondria opened new hypotheses about its role in pregnancy. However, much further work is needed to understand the importance of these proteins in human pregnancies.

The ERK5/NF-κB signaling pathway targets endometrial cancer proliferation and survival.

Endometrial cancer (EC) is the most common type of gynecologic cancer in women of developed countries. Despite surgery combined with chemo-/radiotherapy regimens, overall survival of patients with high-risk EC tumors is poor, indicating a need for novel therapies. The MEK5-ERK5 pathway is activated in response to growth factors and to different stressors, including oxidative stress and cytokines. Previous evidence supports a role for the MEK5-ERK5 pathway in the pathology of several cancers. We investigated the role of ERK5 in EC. In silico analysis of the PanCancer Atlas dataset showed alterations in components of the MEK5-ERK5 pathway in 48% of EC patients. Here, we show that ERK5 inhibition or silencing decreased EGF-induced EC cell proliferation, and that genetic deletion of MEK5 resulted in EC impaired proliferation and reduced tumor growth capacity in nude mice. Pharmacologic inhibition or ERK5 silencing impaired NF-kB pathway in EC cells and xenografts. Furthermore, we found a positive correlation between ERK5 and p65/RELA protein levels in human EC tumor samples. Mechanistically, genetic or pharmacologic impairment of ERK5 resulted in downregulation of NEMO/IKKγ expression, leading to impaired p65/RELA activity and to apoptosis in EC cells and xenografts, which was rescued by NEMO/IKKγ overexpression. Notably, ERK5 inhibition, MEK5 deletion or NF-kB inhibition sensitized EC cells to standard EC chemotherapy (paclitaxel/carboplatin) toxicity, whereas ERK5 inhibition synergized with paclitaxel to reduce tumor xenograft growth in mice. Together, our results suggest that the ERK5-NEMO-NF-κB pathway mediates EC cell proliferation and survival. We propose the ERK5/NF-κB axis as new target for EC treatment.

Ruxolitinib as a treatment strategy for SARS-CoV-2 pneumonia: clinical experience in a real-world setting.

INTRODUCTION: Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) infection is characterised by a viral phase and a severe pro-inflammatory phase. The inhibition of the JAK/STAT pathway limits the pro-inflammatory state in moderate to severe COVID-19. METHODOLOGY: We analysed the data obtained by an observational cohort of patients with SARS-CoV-2 pneumonia treated with ruxolitinib in 22 hospitals of Mexico. The applied dose was determined based on physician's criteria. The benefit of ruxolitinib was evaluated using the 8-points ordinal scale developed by the NIH in the ACTT1 trial. Duration of hospital stay, changes in pro-inflammatory laboratory values, mortality, and toxicity were also measured. RESULTS: A total of 287 patients were reported at 22 sites in Mexico from March to June 2020; 80.8% received ruxolitinib 5 mg BID and 19.16% received ruxolitinib 10 mg BID plus standard of care. At beginning of treatment, 223 patients were on oxygen support and 59 on invasive ventilation. The percentage of patients on invasive ventilation was 53% in the 10 mg and 13% in the 5 mg cohort. A statistically significant improvement measured as a reduction by 2 points on the 8-point ordinal scale was described (baseline 5.39 ± 0.93, final 3.67± 2.98, p = 0.0001). There were 74 deaths. Serious adverse events were presented in 6.9% of the patients. CONCLUSIONS: Ruxolitinib appears to be safe in COVID-19 patients, with clinical benefits observed in terms of decrease in the 8-point ordinal scale and pro-inflammatory state. Further studies must be done to ensure efficacy against mortality.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization.

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin-1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP-2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress.

Autophagy is a highly conserved intracellular process that preserves cellular homeostasis by mediating the lysosomal degradation of virtually any component of the cytoplasm. Autophagy is a key instrument of cellular response to several stresses, including endoplasmic reticulum (ER) stress. Cancer cells have developed high dependency on autophagy to overcome the hostile tumor microenvironment. Thus, pharmacological activation or inhibition of autophagy is emerging as a novel antitumor strategy. ERK5 is a novel member of the MAP kinase family that is activated in response to growth factors and different forms of stress. Recent work has pointed ERK5 as a major player controlling cancer cell proliferation and survival. Therefore small-molecule inhibitors of ERK5 have shown promising therapeutic potential in different cancer models. Here, we report for the first time ERK5 as a negative regulator of autophagy. Thus, ERK5 inhibition or silencing induced autophagy in a panel of human cancer cell lines with different mutation patterns. As reported previously, ERK5 inhibitors (ERK5i) induced apoptotic cancer cell death. Importantly, we found that autophagy mediates the cytotoxic effect of ERK5i, since ATG5-/- autophagy-deficient cells viability was not affected by these compounds. Mechanistically, ERK5i stimulated autophagic flux independently of the canonical regulators AMPK or mTORC1. Moreover, ERK5 inhibition resulted in ER stress and activation of the Unfolded Protein Response (UPR) pathways. Specifically, ERK5i induced expression of the ER luminal chaperone BiP (a hallmark of ER stress), the UPR markers CHOP and ATF4, and the spliced form of XBP1. Pharmacological inhibition of UPR with chemical chaperone TUDC, or ATF4 silencing, resulted in impaired ERK5i-mediated UPR, autophagy and cytotoxicity. Overall, our results suggest that ERK5 inhibition induces autophagy-mediated cancer cell death by activating ER stress. Since ERK5 inhibition sensitizes cancer cells and tumors to chemotherapy, future work will determine the relevance of UPR and autophagy in the combined use of chemotherapy and ERK5i to tackle Cancer.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

[Carbapenamase OXA-23 like-producing Acinetobacter baumanii epidemic outbreak in a hospitalization unit.] / Brote epidémico por Acinetobacter baumanii productor de carbapenamasa OXA-23 like en una unidad de hospitalización.

OBJECTIVE: Despite its great ubiquity and morbidity and mortality, the scientific evidence on the hospital control of multiresistant Acinetobacter baumanii (ABMR) outside the Intensive Care Units in Spain is scarce. The objective was to describe an epidemic outbreak by MRAB and analyze the effectiveness of the actions carried out. METHODS: Prospective observational study of admitted-rotated patients in a multipathological control at the University Hospital of Guadalajara, Spain, during the outbreak (September 20-November 3, 2017); using Mambrino Electronic Health Record. A genetic study of the resistance mechanism and molecular characterization of the strains were carried out. Frequency measurements were estimated, with subsequent comparative analysis of cases vs controls. RESULTS: The median age of the study population (N=138) was 83.2 years (Interquartile Range [IR]=69.7-90.1). There were 3 cases of ABMR infection among them. Thirteen percent required issolation, 17% because of MRAB. The MRAB incidence was 2.2 cases/100 admitted-rotated (mortality rate=33%). The excess stay for cases was 17±4.3 (95%CI=8.5-25.6), with an incidence density of 3 cases/103 days. The responsible strain was carbapenemase OXA-23. We found a single case in the colonization study of contacts. No MRAB was isolated from environmental samples. CONCLUSIONS: Along with epidemiological research, coordination and compliance with precautions; prompt identification and management of an outbreak are crucial to minimize the colonization pressure and to stop dissemination.

OBJETIVO: Pese a su gran ubicuidad y morbimortalidad, la evidencia científica sobre el control hospitalario de Acinetobacter baumanii multirresistente (ABMR) fuera de las Unidades de Cuidados Intensivos en España es escasa. El objetivo fue describir un brote epidémico por ABMR y analizar la efectividad del manejo. METODOS: Se realizó un estudio observacional retrospectivo de los ingresados-rotados (N=138) en un control de pluripatológicos del Hospital Universitario de Guadalajara (España), durante el brote (20 septiembre-3 noviembre de 2017), utilizando la Historia Clínica Electrónica Mambrino. Se realizó un estudio genético del mecanismo de resistencia y caracterización molecular de cepas. Se estimaron medidas de frecuencia y análisis comparativo de casos respecto a controles. RESULTADOS: La mediana de edad de la población expuesta fue de 83,2 años (Rango Intercuartílico [RI]=69,7-90,1). Hubo entre ellos tres casos de infección por ABMR. Requirieron aislamiento un 13% de los ingresados-rotados, el 17% por ABMR. La incidencia de ABMR fue de 2,2 por cada 100 ingresados-rotados (tasa de mortalidad=33%). El exceso de estancia de los casos fue de 17±4,3 días (IC95%=8,5-25,6), con una densidad de incidencia de 3/103 días. La cepa responsable fue carbapenemasa OXA-23. En el estudio de colonización de contactos se evidenció un caso. El estudio ambiental resultó negativo. CONCLUSIONES: Junto a la investigación epidemiológica, la coordinación y el cumplimiento de precauciones, la prontitud en la identificación y gestión del brote son determinantes para minimizar la presión de colonización y frenar la cadena de diseminación.

The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.

ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased DDIT3 and TRIB3 mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ9-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.

Assessing the Role of Uric Acid as a Predictor of Preeclampsia.

We assessed the diagnostic utility of uric acid for the prediction of preeclampsia. An observational prospective approach was carried out during 2014. Preeclamptic women were classified into 4 groups accordingly to the onset of preeclampsia and the presence of intrauterine growth restriction (IUGR). Serum uric acid levels, urea, and creatinine were measured. Receiver operating curves (ROC) of the uric acid levels ratio (UAr) between a dosage before and after the 20th week of gestation were performed. One thousand two hundred and ninety-third pregnant women were enrolled in this study. Eight hundred ten had non-complicated pregnancies, 40 preeclampsia, 33 gestational hypertension, and 20 IUGR without preeclampsia. Uric acid significantly raised after 20 weeks of gestation in women who develop preeclampsia before 34 weeks (Group A) or in those who develop preeclampsia after 37 weeks associated with IUGR (Group C). In women who develop preeclampsia after 34 weeks without IUGR (Groups B and D), uric acid increased after the 30th week of gestation. In all groups, UAr was greater than 1.5. In gestational hypertension, UAr was superior to 1.5 toward the end of gestation, while in IUGR without preeclampsia, the behavior of serum uric acid was similar to non-complicated pregnancies. In all cases, urea and creatinine showed normal values, confirming that patients had no renal compromise. ROC area was 0.918 [95% confidence interval (CI): 0.858-0.979) for the preeclampsia group and 0.955 (95% CI: 0.908-1.000) for Group A. UAr at a cut-off point ≥1.5 had a very low positive predictive value, but a high negative predictive value of 99.5% for preeclampsia and it reached 100% for Group A. Thus, a UAr less than 1.5 may be a helpful parameter with a strong exclusion value and high sensitivity for those women who are not expected to develop preeclampsia. Additionally, this low-cost test would allow for better use of resources in developing countries.

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.

Discovery of a selective inhibitor of doublecortin like kinase 1.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

SUMOylation Is Required for ERK5 Nuclear Translocation and ERK5-Mediated Cancer Cell Proliferation.

The MAP kinase ERK5 contains an N-terminal kinase domain and a unique C-terminal tail including a nuclear localization signal and a transcriptional activation domain. ERK5 is activated in response to growth factors and stresses and regulates transcription at the nucleus by either phosphorylation or interaction with transcription factors. MEK5-ERK5 pathway plays an important role regulating cancer cell proliferation and survival. Therefore, it is important to define the precise molecular mechanisms implicated in ERK5 nucleo-cytoplasmic shuttling. We previously described that the molecular chaperone Hsp90 stabilizes and anchors ERK5 at the cytosol and that ERK5 nuclear shuttling requires Hsp90 dissociation. Here, we show that MEK5 or overexpression of Cdc37-mechanisms that increase nuclear ERK5-induced ERK5 Small Ubiquitin-related Modifier (SUMO)-2 modification at residues Lys6/Lys22 in cancer cells. Furthermore, mutation of these SUMO sites abolished the ability of ERK5 to translocate to the nucleus and to promote prostatic cancer PC-3 cell proliferation. We also show that overexpression of the SUMO protease SENP2 completely abolished endogenous ERK5 nuclear localization in response to epidermal growth factor (EGF) stimulation. These results allow us to propose a more precise mechanism: in response to MEK5 activation, ERK5 SUMOylation favors the dissociation of Hsp90 from the complex, allowing ERK5 nuclear shuttling and activation of the transcription.

Intact caveolae are required for proper extravillous trophoblast migration and differentiation.

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-ß-cyclodextrin (MßCD) to disrupt caveolae. We found that after MßCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MßCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.

Osmotic stress induces apoptosis in extravillous trophoblast cells. Role of TRPV-1.

In different tissues hyperosmolarity induces cell differentiation. Nevertheless an exacerbated hyperosmolar stress alters the normal cellular development. The transient receptor potential vanilloid 1 (TRPV-1) is a non-selective cation channel that is activated by hyperosmolarity and participates in many cellular processes. TPRV-1 is expressed in human placenta at term. Here, we evaluated the expression of TRPV-1 in first trimester extravillous trophoblast cells and its participation in the survival of these cells exposed to hyperosmolar stress. Our results showed that hyperosmolar stress up-regulates the expression of TRPV-1 and induces the apoptosis in Swan 71 cells. In addition, the inhibition of TRPV-1 abrogates the apoptotic events.

Crosstalk Between Nitric Oxide and Endocannabinoid Signaling Pathways in Normal and Pathological Placentation.

Endocannabinoids are a group of endogenous lipid mediators that act as ligands of cannabinoid and vanilloid receptors, activating multiple signal transduction pathways. Together with enzymes responsible for their synthesis and degradation, these compounds constitute the endocannabinoid system (ECS), which is involved in different physiological processes in reproduction. The placenta, which is essential for the success of gestation and optimal fetal growth, undergoes constant tissue remodeling. ECS members are expressed in trophoblast cells, and current evidence suggests that this system is involved in placental development, apoptosis, and syncytialization. Impairment of endocannabinoid signaling has been associated with several pathological conditions such as intrauterine growth restriction and preeclampsia. Both clinical entities are characterized by dysregulation on vascular perfusion where nitrergic system performs a pivotal role. Nitric oxide (NO) is a potent local vasodepressor that exerts a critical role in the regulation of hemodynamic flow, contributing to the maintenance of low vascular resistance in the feto-placental circulation. NO production could be affected by different factors and growing evidence suggests that the endocannabinoid mediators may regulate nitrergic signaling. Herein, we review emerging knowledge supporting ECS-mediated regulation of NO production in normal placentation. Finally, we discuss how alterations in these systems could affect homoeostasis and contribute to the occurrence of placental-mediated pregnancy complications. Given the impact on women and perinatal heath, we will focus on current knowledge regarding the effects of ECS on nitrergic system in normal and pathological placentation.

Oxygen regulation of aquaporin-4 in human placenta.

RESEARCH QUESTION: We recently reported that blocking of placental aquaporins (AQP) abrogates the apoptotic response of the trophoblast. As trophoblast apoptosis is exacerbated in pre-eclampsia, we hypothesized that changes in AQP in these placentae may trigger programmed cell death. We analysed AQP4 expression in pre-eclamptic placentae and its regulation by oxygen tension. DESIGN: AQP4 expression was studied in placentae from non-pathological and pre-eclamptic pregnancies by reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence and immunohistochemistry. Explants from non-pathological placentae were cultured in normoxia, hypoxia, hypoxia-reoxygenation and CoCl2. AQP4 expression was investigated by RT-PCR and Western blot. Hypoxia responsive elements sites on AQP4 promotor were investigated by in-silico analysis. AQP4 degradation was studied in the presence of proteosomal and lysosomal inhibitors. RESULTS: AQP4 protein expression was weakly detectable in pre-eclamptic placentae, but its mRNA was elevated compared with non-pathological placentae. In non-pathological explants cultured in hypoxia, AQP4 mRNA and protein were increased compared with placentae cultured in ambient oxygen but decreased after reoxygenation. Incubation with CoCl2, that stabilizes hypoxia inducible factor (HIF)-1α, also increased AQP4 levels. In-silico analysis showed three putative binding sites for HIF-1α in AQP4 promotor. CONCLUSIONS: Oxygen may regulate AQP4 expression in human placenta, possibly through HIF-1α. Therefore, the decrease in AQP4 throughout pregnancy, previously reported, is consistent with changes in HIF-1α, and suggests that AQP4 might have a crucial role during placentation. Therefore, the abnormal expression of AQP4 may be involved in the cause of pre-eclampsia, but it does not seem to take part in the apoptotic events.

New Insights Into the Role of Placental Aquaporins and the Pathogenesis of Preeclampsia.

Accumulated evidence suggests that an abnormal placentation and an altered expression of a variety of trophoblast transporters are associated to preeclampsia. In this regard, an abnormal expression of AQP3 and AQP9 was reported in these placentas. Recent data suggests that placental AQPs are not only water channel proteins and that may participate in relevant processes required for a normal placental development, such as cell migration and apoptosis. Recently we reported that a normal expression of AQP3 is required for the migration of extravillous trophoblast (EVT) cells. Thus, alterations in this protein might lead to an insufficient transformation of the maternal spiral arteries resulting in fluctuations of oxygen tension, a potent stimulus for oxidative damage and trophoblast apoptosis. In this context, the increase of oxygen and nitrogen reactive species could nitrate AQP9, producing the accumulation of a non-functional protein affecting the survival of the villous trophoblast (VT). This may trigger the exacerbated release of apoptotic VT fragments into maternal circulation producing the systemic endothelial dysfunction underlying the maternal syndrome. Therefore, our hypothesis is that the alteration in the expression of placental AQPs observed at the end of gestation may take place during the trophoblast stem cell differentiation, disturbing both EVT and VT cells development, or during the VT differentiation and turnover. In both situations, VT is affected and at last the maternal vascular system is activated leading to the clinical manifestations of preeclampsia.