Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy.

BACKGROUND: Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood. METHODS: The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer. RESULTS: In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response. CONCLUSION: HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors.

Aula invertida y aula virtual en la formación del Técnico Superior en neurofisiología clínica

Introducción: En las ciencias médicas son diversas las investigaciones asociadas a la modalidad virtual, sobre todo durante la pandemia por COVID-19. Sin embargo, en el campo de la neurofisiología clínica existen pocas experiencias descritas. Objetivo: Describir los resultados del desarrollo virtual de las asignaturas Historia de la Neurociencia y la Neurofisiología en Cuba e Introducción a la Metodología de la Investigación Científica, mediante el aula invertida y el aula virtual en la formación del Técnico Superior en neurofisiología clínica en La Habana. Métodos: Estudio descriptivo de corte transversal y no experimental. Se adecuaron las asignaturas en modalidad virtual, al aplicar un modelo constituido por cuatro fases: preparación, análisis y diseño, desarrollo e implementación, y evaluación. Resultados: Se preparó al claustro en materia de educación virtual. Se diseñaron estrategias didácticas para la virtualización del proceso de enseñanza-aprendizaje, mediante la aplicación del aula invertida y el aula virtual. En la fase de evaluación se obtuvieron valores altos al calcular los indicadores de promoción y calidad, y se aplicó una encuesta a los estudiantes. Para ambas asignaturas predominaron los valores de alta satisfacción en los criterios de la encuesta, excepto en "Formas de evaluaciones" y "Distribución del tiempo en el aprendizaje", con mayores valores de satisfacción media. Conclusión: Los resultados obtenidos en el tránsito a la modalidad virtual cumplieron con los objetivos propuestos en el programa de las asignaturas, por lo que fue posible la enseñanza no presencial mediante el aula invertida y el aula virtual.

Introduction: In medical sciences, there are several pieces of research associated with the virtual modality, especially during the COVID-19 pandemic. However, in the field of clinical neurophysiology, few experiences have been described. Objective: To describe the outcomes of virtually developing the subjects History of Neuroscience and Neurophysiology in Cuba and Introduction to the Methodology of Scientific Research, by means of the inverted classroom and the virtual classroom in the training of higher-degree associates in clinical neurophysiology in Havana. Methods: A descriptive, cross-sectional and nonexperimental study was carried out. The subjects were adapted to the virtual modality by applying a model consisting of four phases: preparation, analysis and design, development and implementation, and evaluation. Results: The faculty was trained in virtual education. Didactic strategies were designed for the virtualization of the teaching-learning process, through the application of the inverted classroom and the virtual classroom. In the evaluation phase, high values were obtained when calculating the promotion and quality indicators, and a student survey was applied. For both subjects, high satisfaction values predominated in the survey criteria, except for evaluations forms and distribution of time for learning, with higher values of the medium satisfaction. Conclusions: The outcomes obtained in the transition to the virtual modality complied with the objectives set in the syllabus of the subjects, making possible non-face-to-face teaching by means of the inverted classroom and the virtual classroom.

ZMYM2 controls human transposable element transcription through distinct co-regulatory complexes.

ZMYM2 is a zinc finger transcriptional regulator that plays a key role in promoting and maintaining cell identity. It has been implicated in several diseases such as congenital anomalies of the kidney where its activity is diminished and cancer where it participates in oncogenic fusion protein events. ZMYM2 is thought to function through promoting transcriptional repression and here we provide more evidence to support this designation. Here we studied ZMYM2 function in human cells and demonstrate that ZMYM2 is part of distinct chromatin-bound complexes including the established LSD1-CoREST-HDAC1 corepressor complex. We also identify new functional and physical interactions with ADNP and TRIM28/KAP1. The ZMYM2-TRIM28 complex forms in a SUMO-dependent manner and is associated with repressive chromatin. ZMYM2 and TRIM28 show strong functional similarity and co-regulate a large number of genes. However, there are no strong links between ZMYM2-TRIM28 binding events and nearby individual gene regulation. Instead, ZMYM2-TRIM28 appears to regulate genes in a more regionally defined manner within TADs where it can directly regulate co-associated retrotransposon expression. We find that different types of ZMYM2 binding complex associate with and regulate distinct subclasses of retrotransposons, with ZMYM2-ADNP complexes at SINEs and ZMYM2-TRIM28 complexes at LTR elements. We propose a model whereby ZMYM2 acts directly through retrotransposon regulation, which may then potentially affect the local chromatin environment and associated coding gene expression.

Neuronal Prosurvival Role of Ceramide Synthase 2 by Olidogendrocyte-to-Neuron Extracellular Vesicle Transfer.

Ageing is associated with notorious alterations in neurons, i.e., in gene expression, mitochondrial function, membrane degradation or intercellular communication. However, neurons live for the entire lifespan of the individual. One of the reasons why neurons remain functional in elderly people is survival mechanisms prevail over death mechanisms. While many signals are either pro-survival or pro-death, others can play both roles. Extracellular vesicles (EVs) can signal both pro-toxicity and survival. We used young and old animals, primary neuronal and oligodendrocyte cultures and neuroblastoma and oligodendrocytic lines. We analysed our samples using a combination of proteomics and artificial neural networks, biochemistry and immunofluorescence approaches. We found an age-dependent increase in ceramide synthase 2 (CerS2) in cortical EVs, expressed by oligodendrocytes. In addition, we show that CerS2 is present in neurons via the uptake of oligodendrocyte-derived EVs. Finally, we show that age-associated inflammation and metabolic stress favour CerS2 expression and that oligodendrocyte-derived EVs loaded with CerS2 lead to the expression of the antiapoptotic factor Bcl2 in inflammatory conditions. Our study shows that intercellular communication is altered in the ageing brain, which favours neuronal survival through the transfer of oligodendrocyte-derived EVs containing CerS2.

NAD pool as an antitumor target against cancer stem cells in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that affect different anatomical locations. Despite this heterogeneity, HNSCC treatment depends on the anatomical location, TNM stage and resectability of the tumor. Classical chemotherapy is based on platinum-derived drugs (cisplatin, carboplatin and oxaliplatin), taxanes (docetaxel, paclitaxel) and 5-fluorouracil1. Despite advances in HNSCC treatment, the rate of tumor recurrence and patient mortality remain high. Therefore, the search for new prognostic identifiers and treatments targeting therapy-resistant tumor cells is vital. Our work demonstrates that there are different subgroups with high phenotypic plasticity within the CSC population in HNSCC. CD10, CD184, and CD166 may identify some of these CSC subpopulations with NAMPT as a common metabolic gene for the resilient cells of these subpopulations. We observed that NAMPT reduction causes a decrease in tumorigenic and stemness properties, migration capacity and CSC phenotype through NAD pool depletion. However, NAMPT-inhibited cells can acquire resistance by activating the NAPRT enzyme of the Preiss-Handler pathway. We observed that coadministration of the NAMPT inhibitor with the NAPRT inhibitor cooperated inhibiting tumor growth. The use of an NAPRT inhibitor as an adjuvant improved NAMPT inhibitor efficacy and reduced the dose and toxicity of these inhibitors. Therefore, it seems that the reduction in the NAD pool could have efficacy in tumor therapy. This was confirmed by in vitro assays supplying the cells with products of inhibited enzymes (NA, NMN or NAD) and restoring their tumorigenic and stemness properties. In conclusion, the coinhibition of NAMPT and NAPRT improved the efficacy of antitumor treatment, indicating that the reduction in the NAD pool is important to prevent tumor growth.

Validación de un test de detección temprana de dificultades de la lectoescritura en Ecuador. Validación de un test de riesgo de dificultades / Validation of a test for early detection of literacy difficulties in Ecuador. Validation of a hardship risk test

Introducción: Las dificultades del aprendizaje son las alteraciones de mayor presencia en las aulas escolares y sus indicadores pueden diagnosticarse y prevenirse desde edades tempranas. El objetivo de esta investigación fue validar el Test para la detección temprana de las dificultades en el aprendizaje de la lectura y escritura. Métodos: El enfoque de la investigación fue cuantitativo, descriptivo y de corte transversal. Se utilizó la validez de constructo acorde con la propuesta original del test y de fiabilidad a través del Alpha de Cronbach en una muestra de 501 niños ecuatorianos de cuatro años. Resultados: La validación del instrumento evidencia una moderada correlación entre las sub-tareas y alta correlación entre las sub-tareas y el puntaje total. La fiabilidad es buena, α= 0.71, muy próxima a la de la población española α= 0.73. Por lo que, la prueba puede ser utilizada en el contexto ecuatoriano en su versión original, adecuando en las instrucciones dos palabras a la realidad lingüística del país y para la calificación los puntos de corte de dificultad. Conclusión: Considerando su valor y fácil aplicación se recomienda el uso de la prueba de lectura en contextos educativos y de salud.

Introduction: Learning difficulties are the alterations with the most significant presence in school classrooms, and their indicators can be diagnosed and prevented early. This research aimed to validate the test for the early detection of difficulties in learning to read and write. Methods: The research approach was quantitative, descriptive, and cross-sectional. Construct validity was used according to the original proposal of the test and reliability through Cronbach's alpha in a sample of 501 four-year-old Ecuadorian children. Results: The validation of the instrument shows a moderate correlation between the subtasks and a high correlation between the subtasks and the total score. The reliability is good, α = 0.71, very close to that of the Spanish population α = 0.73. Therefore, the test can be used in the Ecuadorian context in its original version, adapting two words in the instructions to the linguistic reality of the country and for the qualification of the cutoff points of difficulty. Conclusion: With the easy application of the "test of reading" in 4-year-old children, the authors recommended its application for the identification of dyslexia and phonological processing deficits in school children in Ecuador. The reading test's validity allows its application at a regional level.

3D and organoid culture in research: physiology, hereditary genetic diseases and cancer.

In nature, cells reside in tissues subject to complex cell-cell interactions, signals from extracellular molecules and niche soluble and mechanical signaling. These microenvironment interactions are responsible for cellular phenotypes and functions, especially in normal settings. However, in 2D cultures, where interactions are limited to the horizontal plane, cells are exposed uniformly to factors or drugs; therefore, this model does not reconstitute the interactions of a natural microenvironment. 3D culture systems more closely resemble the architectural and functional properties of in vivo tissues. In these 3D cultures, the cells are exposed to different concentrations of nutrients, growth factors, oxygen or cytotoxic agents depending on their localization and communication. The 3D architecture also differentially alters the physiological, biochemical, and biomechanical properties that can affect cell growth, cell survival, differentiation and morphogenesis, cell migration and EMT properties, mechanical responses and therapy resistance. This latter point may, in part, explain the failure of current therapies and affect drug discovery research. Organoids are a promising 3D culture system between 2D cultures and in vivo models that allow the manipulation of signaling pathways and genome editing of cells in a body-like environment but lack the many disadvantages of a living system. In this review, we will focus on the role of stem cells in the establishment of organoids and the possible therapeutic applications of this model, especially in the field of cancer research.

Germline mutation landscape of DNA damage repair genes in African Americans with prostate cancer highlights potentially targetable RAD genes.

In prostate cancer, emerging data highlight the role of DNA damage repair genes (DDRGs) in aggressive forms of the disease. However, DDRG mutations in African American men are not yet fully defined. Here, we profile germline mutations in all known DDRGs (N = 276) using whole genome sequences from blood DNA of a matched cohort of patients with primary prostate cancer comprising of 300 African American and 300 European Ancestry prostate cancer patients, to determine whether the mutation status can enhance patient stratification for specific targeted therapies. Here, we show that only 13 of the 46 DDRGs identified with pathogenic/likely pathogenic mutations are present in both African American and European ancestry patients. Importantly, RAD family genes (RAD51, RAD54L, RAD54B), which are potentially targetable, as well as PMS2 and BRCA1, are among the most frequently mutated DDRGs in African American, but not in European Ancestry patients.

BAPST. A Combo of Common Use Drugs as Metabolic Therapy for Cancer: A Theoretical Proposal.

Cancer therapy advances have yet to impact global cancer mortality. One of the factors limiting mortality burden reduction is the high cost of cancer drugs. Cancer drug repurposing has already failed to meet expectations in terms of drug affordability. The three FDA-approved cancer drugs developed under repurposing: all-trans-retinoic acid, arsenic trioxide, and thalidomide do not differ in price from other drugs developed under the classical model. Though additional factors affect the whole process from inception to commercialization, the repurposing of widely used, commercially available, and cheap drugs may help. This work reviews the concept of the malignant metabolic phenotype and its exploitation by simultaneously blocking key metabolic processes altered in cancer. We elaborate on a combination called BAPST, which stands for the following drugs and pathways they inhibit: Benserazide (glycolysis), Apomorphine (glutaminolysis), Pantoprazole (Fatty-acid synthesis), Simvastatin (mevalonate pathway), and Trimetazidine (Fatty-acid oxidation). Their respective primary indications are: • Parkinson's disease (benserazide and apomorphine). • Peptic ulcer disease (pantoprazole). • Hypercholesterolemia (simvastatin). • Ischemic heart disease (trimetazidine). When used for their primary indication, the literature review on each of these drugs shows that they have a good safety profile and lack predicted pharmacokinetic interaction among them. Based on that, we propose that the BAPST regimen merits preclinical testing.

Evaluación del desarrollo de la asignatura virtual Fisiología II en la especialidad Neurofisiología Clínica / Assessment of developing Physiology II as a virtual subject in the specialty of Clinical Neurophysiology

Introducción: La enseñanza mediante la modalidad virtual se adoptó como medida preventiva ante la propagación de la COVID-19. Para la educación médica cubana ha implicado un reto en cuanto a la reorganización de los programas de estudio. Con este propósito, se rediseño e implementó la asignatura Fisiología II en modalidad virtual, para garantizar la continuidad en la formación de residentes en Neurofisiología Clínica. Objetivo: Evaluar el desarrollo de la asignatura Fisiología II en su modalidad virtual desde la perspectiva de los estudiantes de la especialidad Neurofisiología Clínica. Métodos: Estudio exploratorio, no experimental y transversal. Mediante un cuestionario de preguntas cerradas y una abierta (aspectos positivos y negativos), se evaluó el desarrollo de la asignatura en modalidad virtual en relación con las variables: programa de la asignatura, desempeño de los profesores, escenario de las plataformas y soporte tecnológico. Resultados: La asignatura Fisiología II en modalidad virtual tuvo una alta aceptación por parte de los estudiantes. Los aspectos identificados como positivos fueron los relacionados con la gestión personal del tiempo, y la aplicabilidad y estructura de la asignatura; mientras que los negativos se orientaron hacia problemas de conectividad de internet y el entorno de estudio. Conclusiones: Las exitosas experiencias obtenidas en el desarrollo de la asignatura virtual Fisiología II sientan las bases para el empleo de este tipo de diseño en la especialidad de Neurofisiología Clínica y la posibilidad de extenderlo a otras asignaturas del plan de estudio(AU)

Introduction: Teaching through virtual modality was adopted as a preventive measure against the COVID-19 spread. For Cuban medical education, it has implied a challenge regarding the reorganization of study programs. For this purpose, the subject Physiology II was redesigned and implemented in virtual modality, in order to guarantee continuity in the training of Clinical Neurophysiology residents. Objective: To assess the development of the subject Physiology II in its virtual modality from the perspective of the students of the Clinical Neurophysiology specialty. Methods: Exploratory, nonexperimental and cross-sectional study. Through a questionnaire made up of closed questions and one open question (positive and negative aspects), the development of the subject in virtual modality was assessed in relation to the variables subject syllabus, professors' performance, platform scenario, and technological support. Results: The subject Physiology II in virtual modality had a high acceptance by the students. The aspects identified as positive were those related to individual time management, as well as the subject's applicability and structure; while the negative ones were oriented towards internet connectivity problems and the study environment. Conclusions: The successful experiences obtained in the development of Physiology II as a virtual subject lay the foundations for using this type of design in the specialty of Clinical Neurophysiology and the possibility of extending it to other subjects of the study plan(AU)

DL4-µbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system.

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-µbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αß T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-µbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

The careful control of Polo kinase by APC/C-Ube2C ensures the intercellular transport of germline centrosomes during Drosophila oogenesis.

A feature of metazoan reproduction is the elimination of maternal centrosomes from the oocyte. In animals that form syncytial cysts during oogenesis, including Drosophila and human, all centrosomes within the cyst migrate to the oocyte where they are subsequently degenerated. The importance and the underlying mechanism of this event remain unclear. Here, we show that, during early Drosophila oogenesis, control of the Anaphase Promoting Complex/Cyclosome (APC/C), the ubiquitin ligase complex essential for cell cycle control, ensures proper transport of centrosomes into the oocyte through the regulation of Polo/Plk1 kinase, a critical regulator of the integrity and activity of the centrosome. We show that novel mutations in the APC/C-specific E2, Vihar/Ube2c, that affect its inhibitory regulation on APC/C cause precocious Polo degradation and impedes centrosome transport, through destabilization of centrosomes. The failure of centrosome migration correlates with weakened microtubule polarization in the cyst and allows ectopic microtubule nucleation in nurse cells, leading to the loss of oocyte identity. These results suggest a role for centrosome migration in oocyte fate maintenance through the concentration and confinement of microtubule nucleation activity into the oocyte. Considering the conserved roles of APC/C and Polo throughout the animal kingdom, our findings may be translated into other animals.

Cutting Edge: TCR-ß Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.

Programa cubano de pesquisa neonatal de la hiperplasia suprarrenal congénita: una realidad. 2005-2014 / Cuban neonatal screening program for congenital adrenal hyperplasia: a reality. 2005­2014

Introducción: la hiperplasia suprarrenal congénita (HSC) es el desorden adrenal más común en la infancia y la causa más frecuente de ambigüedad sexual. La forma clásica, que representa los casos más severos de este déficit, se asocia en un 75 % con pérdida de sal. Por otra parte, en los recién nacidos (RN) del sexo femenino se pueden presentar grados severos de virilización de genitales. Objetivo: presentar los resultados (durante diez años), del Programa Cubano de Pesquisa Neonatal (PN) de la HSC, soportado en un procedimiento inmunoenzimático desarrollado en Cuba. Resultados: en el período de enero 2005 a diciembre 2014, se han estudiado 1 140 882 RN y se detectaron 56 niños con HSC, para una incidencia de 1:20 373 RN. La cobertura del programa se ha incrementado hasta llegar en el año 2013 al 99.34 % de todos los RN cubanos. Conclusiones: la existencia del Programa Cubano de PN de HSC, ha permitido estimar la incidencia e incrementar el conocimiento de esta enfermedad. La PN ha posibilitado el diagnóstico precoz en la variedad perdedora de sal, contribuyendo a la disminución de la mortalidad infantil. El Programa ha favorecido a pacientes con formas virilizantes de la enfermedad, mediante la asignación correcta del sexo

Introduction: Congenital Adrenal Hyperplasia (HSC) is the most common adrenal disorder in childhood and the most frequent cause of sexual ambiguity. The classic form, which represents the most severe causes of this deficit, is associated in 75 % with loss of salt. On the other hand, in the NB of the female sex that present severe degrees of virilization of the genitals. Objective: To present result of the application for ten years of the Cuban Neonatal Research Program of the HSC, supported by an inmmunoenzymatic procedure developed in Cuba. Result: In the period from January 2005 to December 2014, using the UMELISA 17 OH PROGESTERONA NEONATAL, 1 140882 RN were detected, for an incidence of 1:20373 RN. The coverage of the program has been increasing until2013 reaching 99.34 % of all Cuban RN. Conclusion: The existence of the Cuban HSC PN Program, has allowed estimating the incidence and increase knowledge of this disease in our country. PN has made possible the early diagnosis of patients with the salt losing variety, contributing to decrease in infant mortality nationwide. The Program has favored patients with virilizing form of the disease, through the correct assignment of sex

Therapeutic Targeting of Signaling Pathways Related to Cancer Stemness.

The theory of cancer stem cells (CSCs) proposes that the different cells within a tumor, as well as metastasis deriving from it, are originated from a single subpopulation of cells with self-renewal and differentiation capacities. These cancer stem cells are supposed to be critical for tumor expansion and metastasis, tumor relapse and resistance to conventional therapies, such as chemo- and radiotherapy. The acquisition of these abilities has been attributed to the activation of alternative pathways, for instance, WNT, NOTCH, SHH, PI3K, Hippo, or NF-κB pathways, that regulate detoxification mechanisms; increase the metabolic rate; induce resistance to apoptotic, autophagic, and senescence pathways; promote the overexpression of drug transporter proteins; and activate specific stem cell transcription factors. The elimination of CSCs is an important goal in cancer therapeutic approaches because it could decrease relapses and metastatic dissemination, which are main causes of mortality in oncology patients. In this work, we discuss the role of these signaling pathways in CSCs along with their therapeutic potential.

Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness.

BACKGROUND: Ovarian cancer is one of the most common and malignant cancers, partly due to its late diagnosis and high recurrence. Chemotherapy resistance has been linked to poor prognosis and is believed to be linked to the cancer stem cell (CSC) pool. Therefore, elucidating the molecular mechanisms mediating therapy resistance is essential to finding new targets for therapy-resistant tumors. METHODS: shRNA depletion of MYPT1 in ovarian cancer cell lines, miRNA overexpression, RT-qPCR analysis, patient tumor samples, cell line- and tumorsphere-derived xenografts, in vitro and in vivo treatments, analysis of data from ovarian tumors in public transcriptomic patient databases and in-house patient cohorts. RESULTS: We show that MYPT1 (PPP1R12A), encoding myosin phosphatase target subunit 1, is downregulated in ovarian tumors, leading to reduced survival and increased tumorigenesis, as well as resistance to platinum-based therapy. Similarly, overexpression of miR-30b targeting MYPT1 results in enhanced CSC-like properties in ovarian tumor cells and is connected to the activation of the Hippo pathway. Inhibition of the Hippo pathway transcriptional co-activator YAP suppresses the resistance to platinum-based therapy induced by either low MYPT1 expression or miR-30b overexpression, both in vitro and in vivo. CONCLUSIONS: Our work provides a functional link between the resistance to chemotherapy in ovarian tumors and the increase in the CSC pool that results from the activation of the Hippo pathway target genes upon MYPT1 downregulation. Combination therapy with cisplatin and YAP inhibitors suppresses MYPT1-induced resistance, demonstrating the possibility of using this treatment in patients with low MYPT1 expression, who are likely to be resistant to platinum-based therapy.

Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

Innate immune protection from pneumonia virus of mice induced by a novel immunomodulator is prolonged by dual treatment and mediated by macrophages.

Respiratory syncytial virus (RSV) is responsible for a large proportion of acute lower respiratory tract infections, specifically in children. Pneumonia virus of mice (PVM) causes similar lung pathology and clinical disease in rodents, and is therefore an appropriate model of RSV infection. Previously, we demonstrated that a single intranasal dose of P-I-P, a novel immunomodulator composed of the toll-like receptor 3 agonist poly(I:C), an innate defense regulator peptide and a polyphosphazene, confers protection in Balb/c mice for up to 3 days from lethal PVM-15 infection. In the present study a dual intranasal treatment with P-I-P was shown to extend the duration of the protection conferred by P-I-P from PVM-15 challenge. Balb/c mice treated twice with P-I-P showed higher survival rates and milder clinical signs when compared to animals that received a single P-I-P dose. While the mice treated with two consecutive doses of P-I-P experienced some weight loss, they all recovered. The dual P-I-P treatment mediated infiltration of several innate immune cells into the BALF and lung, including alveolar macrophages, neutrophils, and γδ T cells. Partial depletion of alveolar macrophages decreased survival rates and exacerbated clinical signs of mice subjected to the P-I-P dual treatment regime followed by PVM-15 challenge. This suggests that the alveolar macrophage is at least partially responsible for the protection elicited by this novel prophylactic treatment strategy.

Cuestionario de Clasificación de Consumidores de Cigarrillos. Resultados de su aplicación en Cuba, 2017 / Questionnaire for classification of cigarette consumers. Results obtained from its implementation in Cuba, 2017

RESUMEN Introducción: El consumo de tabaco deviene un trastorno adictivo que pone en peligro la salud tanto de los fumadores como de los que lo rodean. Su evaluación adecuada requiere contemplar los factores psicológicos y sociales que lo propician. Objetivo: Describir el proceso de adaptación al contexto cubano del Cuestionario de clasificación de consumidores de cigarrillos y caracterizar la muestra de fumadores estudiada para ello. Material y métodos: La investigación desarrollada incluye dos etapas. La primera, clasifica como un estudio de desarrollo tecnológico y la segunda, consiste en un estudio descriptivo de corte transversal con un diseño cuantitativo. Participaron 5 expertos y 205 fumadores activos seleccionados a partir de un muestreo intencional procedentes de áreas de salud de La Habana y Santiago de Cuba. Resultados: Se desarrolló una versión válida para la población cubana del cuestionario, que contiene las recomendaciones realizadas por los expertos y obtuvo una consistencia interna de 0,7005. El 49.7% de los participantes clasificó como fumadores fuertes y 48.3% como fumadores moderados (de riesgo). Por otra parte 60% de ellos son hombres, 32,7% son jóvenes y 65% son trabajadores. Conclusiones: Se realizó la adaptación lingüística y cultural al contexto cubano del Cuestionario de clasificación de consumidores de cigarrillos, la cual cuenta con un nivel de fiabilidad adecuado. La mayor parte de los sujetos estudiados clasificaron como fumadores fuertes o moderados. Predominaron los del sexo masculino, jóvenes y trabajadores.

ABSTRACT Introduction: Tobacco consumption becomes an addictive disorder that endangers the health of both smokers and those around them. Its proper evaluation requires an analysis of the psychological and social factors that favor it. Objective: To describe the adaptation process of the questionnaire for classification of cigarette consumers within the Cuban context and characterize the sample of smokers under study. Material and Methods: The research includes two stages: the first stage that is classified as a technological development study, and the second one which consists of a descriptive cross-sectional study with a quantitative design. Five experts and 205 active smokers selected from an intentional sample from the health areas of Havana and Santiago de Cuba participated in the study. Results: A valid version of the questionnaire for the Cuban population was developed, which contains the recommendations made by the experts and obtained an internal consistency of 0.7005. It was observed that 49.7% of the participants classified as strong smokers and 48.3% as moderate smokers (at risk). On the other hand, 60% of them are men, 32.7% are young and 65% are workers. Conclusions: Linguistic and cultural adaptation to the Cuban context of the questionnaire for classification of cigarette consumers was carried out, which has an adequate level of reliability. Most of the subjects studied were classified as strong or moderate smokers. The male sex, young and workers predominated in the study.