Vitrificación de ovocitos porcinos y su efecto a nivel estructural en embriones y en el ADN de las células del cúmulo

La vitrificación es una técnica que se utiliza principalmente para criopreservar embriones y gametos femeninos. Esta técnica permite mantener la viabilidad celular, la funcionalidad y el potencial de desarrollo a bajas temperaturas en nitrógeno líquido a -196 ºC. Para ello, se requiere de la adición de agentes crioprotectores (CPAs), que son sustancias que brindan protección celular durante el enfriamiento y el calentamiento. Sin embargo, se ha informado que son potencialmente tóxicos, reduciendo la viabilidad de los ovocitos, la maduración, la fertilización y el desarrollo embrionario (DE), posiblemente alterando la estructura del citoesqueleto celular y de la cromatina (CR). Estudios previos han evaluado los efectos de la vitrificación de manera directa en ovocitos en vesícula germinal (VG), metafase II (MII), cigotos y blastocistos, pero el conocimiento de su impacto en el desarrollo posterior de los embriones es limitado. Otros estudios han evaluado el papel de los microfilamentos (MF) de actina y de la CR, basados en las tasas obtenidas de la fertilización y del DE, pero no la evaluación directa de estas estructuras en embriones producidos a partir de ovocitos inmaduros vitrificados. Por lo tanto, este estudio fue diseñado para evaluar cómo la vitrificación de ovocitos inmaduros porcinos afecta al desarrollo del embrión temprano mediante la evaluación de la distribución de MF de actina y la integridad de la CR. Los resultados demuestran que el daño generado por la vitrificación de ovocitos inmaduros afecta la viabilidad, la maduración, el DE, la distribución de MF de actina y la integridad de la CR observada en embriones tempranos, pero no afectó la fertilización in vitro. Por lo tanto, se sugiere que la vitrificación podría afectan los mecanismos de reparación de los ovocitos en esas estructuras, siendo uno de los mecanismos que explicar las bajas tasas de DE después de la vitrificación. 5203 Por otro lado, la evaluación del daño del ADN generado en las células del cúmulo (CC) después de la vitrificación de los complejos ovocitos-células del cúmulo (COCs) maduros puede considerarse como indicador de la calidad de los ovocitos, ya que estas células juegan un papel importante en la competencia del desarrollo de los ovocitos. Por lo tanto, el segundo objetivo de este estudio fue determinar si la exposición de los COCs maduros a CPAs o a la vitrificación afectan la viabilidad de los ovocitos y de las CC, además si se genera daño al ADN en las CC, afectando la fertilización y el DE. El daño del ADN en las CC se midió utilizando el ensayo cometa alcalino y se expresó como longitud de la cola del cometa (LCC) y el tiempo momento (OTM, por sus siglas en inglés). Los resultados demuestran que la exposición de los ovocitos a los CPAs (grupo toxicidad) o la vitrificación redujo la viabilidad de los ovocitos (75.5 ± 3.69 %, toxicidad; 66.7 ± 4.57 %, vitrificación) y de las CC (32.7 ± 5.85 %, toxicidad; 7.7 ± 2.21 %, vitrificación) en comparación con el control (95.5 % ± 4.04 %, ovocitos; 89 ± 4.24 %, CC). Además, se generó daño en el ADN significativamente mayor expresado como OTM en las CC después de la exposición a CPAs y vitrificación (39 ± 17.41, 33.6 ± 16.69, respectivamente) en comparación con el control (7.4 ± 4.22). Además, las tasas de fertilización y de DE también disminuyeron después de la exposición a CPAs (35.3 ± 16.65 %, 22.6 ± 3.05 %, respectivamente) y a la vitrificación (32.3 ± 9.29 %, 20 ± 1 %, respectivamente). Este estudio demuestra que la exposición de los ovocitos a los CPAs o a la vitrificación redujo la viabilidad de los ovocitos y de las CC y generó daños en el ADN de las CC, lo que afectó las tasas de fertilización y de DE. Estos hallazgos permitirán comprender algunos de los mecanismos de daño de los ovocitos tras la vitrificación que comprometen su capacidad de desarrollo, así como la búsqueda de nuevas estrategias de vitrificación para incrementar las tasas de fecundación y de DE preservando la integridad de las CC.Vitrification is mainly used to cryopreserve embryos and female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at -196 ºC. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos, but did not affect in vitro fertilization. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification. On the other hand, the evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69 %, toxicity; 66.7 ± 4.57 %, vitrification) and cumulus cells viability (32.7 ± 5.85 %, toxicity; 7.7 ± 2.21 %, vitrification) compared to control (95.5 ± 4.04 %, oocytes; 89 ± 4.24 %, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65 %, 22.6 ± 3.05 %, respectively) and vitrification (32.3 ± 9.29 %, 20 ± 1 %, respectively). This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells

Vitrificación de ovocitos porcinos y su efecto en la distribución de microfilamentos y cromatina durante el desarrollo embrionario temprano

La vitrificación es una técnica de criopreservación empleada principalmente en gametos. Esta técnica tiene como finalidad mantener la viabilidad celular, su funcionalidad y su potencial de desarrollo a -196°C. Durante este proceso se requiere de la adición de agentes crioprotectores (CPAs), que son sustancias que confieren protección a las células durante el proceso. La selección de la técnica de vitrificación adecuada; depende de la especie animal, el tipo celular y la naturaleza de los CPAs que se utilizan. La toxicidad producida por el uso de los CPAs durante la vitrificación puede causar alteraciones en los microfilamentos (MF) y en la cromatina (CR), esto puede repercutir en la viabilidad, la maduración, la fertilización y el desarrollo embrionario (DE). En estudios previos, se ha evaluado el efecto de la vitrificación en ovocitos en estados de VG y MII, cigotos y blastocistos en la misma etapa de desarrollo en la que fueron vitrificados. Además, estos estudios atribuyeron el papel de los MF y la CR a la disminución de la fertilización y la producción embrionaria, pero no se evaluó la distribución de estas estructuras. Por lo que en este estudio se examinó de qué manera la vitrificación de ovocitos porcinos inmaduros afecta a la distribución de los MF y la CR, así como su repercusión en el DE temprano. La vitrificación, produjo alteraciones en la distribución de la actina cortical (AC) y desarreglo en la compactación de la CR en los embriones, por lo que la fertilización y el DE temprano disminuyeronThe vitrification technique is a type of cryopreservation used mainly in gametes. This technique alows to maintain cell viability, functionality and development potential at -196 ° C. During this technique, the addition of cryoprotective agents (CPAs) is required, which are substances that confer protection to the cells during cooling and warming. Also, the animal species, the cell type and the nature of the CPAs that are used to select the appropriate vitrification technique depends on its success. The toxicity produced by the CPAs during vitrification can cause alterations in microfilaments (MF) and chromatin (CR), also affecting cell viability, maturation, fertilization and embryo development (ED). Previous studies, have evaluated the vitrification effects on oocytes in VG and MII stages, zygotes and blastocysts in the same stage of development in which they were vitrified. In addition, these studies evaluated the role of MF and CR based on the rate of fertilization and embryo production, but not on the distribution of these structures. Therefore, this study was designed to evaluate how the vitrification of immature porcine oocytes affects the distribution of MF and CR, as well as its repercussion in the early ED. In the present study, alterations in the distribution of cortical actin (CA) and disarrangement in the compaction of CR in embryos produced from vitrified oocytes were obtained, reducing the fertilization and early ED rate

Network Traffic Behavioral Analytics for Detection of DDoS Attacks

As more organizations and businesses in different sectors are moving to a digital transformation, there is a steady increase in malware, facing data theft or service interruptions caused by cyberattacks on network or application that impact their customer experience. Bot and Distributed Denial of Service (DDoS) attacks consistently challenge every industry relying on the internet. In this paper, we focus on Machine Learning techniques to detect DDoS attack in network communication flows using continuous learning algorithm that learns the normal pattern of network traffic, behavior of the network protocols and identify a compromised network flow. Detection of DDoS attack will help the network administrators to take immediate action and mitigate the impact of such attacks. DDoS attacks are costing enterprises anywhere between $50,000 to$2.3 million per year. We performed experiments with Intrusion Detection Evaluation Dataset (CICIDS2017) available from Canadian Institute for Cybersecurity to detect anomalies in network traffic. We use flow based traffic characteristics to analyze the difference in pattern between normal vs anomaly packet.We evaluate several supervised classification algorithms using metrics like maximum detection accuracy, lowest false negatives prediction, time taken to train and run. We prove that decision tree based Random Forest is the most promising algorithm whereas Dense Neural network performs equally well on certain DDoS types but require more samples to improve the accuracy of low sampled attacks

Impact of Formative Childhood and Adolescent Experiences in Latinx Children of Immigrants Adulthood: Analysis of Educational, Health, and Social Implications

As the rates of immigration rise within the United States, it is essential to discuss and bring awareness to the neglect and discrimination that immigrants and subsequently the children of immigrants face within the nation. We know about the journey of immigrants and the effects of such but what about their children? Those who did not specifically make the travel to a foreign country but had the “privilege” to be born there? The aim of this study is to investigate the impacts of children of immigrants’ experience in their childhood with this identity and their potential effects into their adulthood by looking into their education, health, and social life. Through a series of online interviews (n=13) with Latinx children of immigrants around the age of 18-30, this thesis found effects in all three areas. For education, participants in their childhood had less school activity participation, language barriers, and differing peer engagement which later affected their perspective and view on education creating pressure to succeed. In regard to health, in their childhood there was a buildup of stress and responsibilities that created elevated anxieties and learned feelings of privilege that as adults they continue to feel yet are working to unlearn. Lastly, in their social life participants explained that in their childhood they missed out on varying social events and yearned to conform to American standards, and as adults now they are working up to catch up to peers while simultaneously embracing their immigrant and Latinx identity

The FOXO1 inhibitor AS1842856 triggers apoptosis in glioblastoma multiforme and basal-like breast cancer cells

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are poor-prognosis cancers that lack effective targeted therapies and harbor embryonic stem gene expression signatures. Recently, our group and others found that forkhead box transcription factor FOXO1 promotes stem gene expression in BBC and GBM cell lines. Given the critical role of cancer stem cells in promoting cancer progression, we examined the impact of FOXO1 inhibition with AS1842856 (a cell-permeable small molecule that directly binds to unphosphorylated FOXO1 protein to block transcriptional regulation) on BBC and GBM cell viability. We treated a set of BBC and GBM cancer cell lines with increasing concentrations of AS1842856 and found reduced colony formation. Treatment of BBC and GBM cancer cells with AS1842856 led to increases in FAS (FAS cell surface death receptor) and BIM (BCL2L11) gene expression, as well as increased positivity for markers for apoptosis such as annexin V and propidium iodide. Treatment with another FOXO1 inhibitor AS1708727 or FOXO1 RNAi also led to FAS induction. This work is the first to show that targeting BBC and GBM with FOXO1 inhibition leads to apoptosis. These novel findings may ultimately expand the repertoire of therapies for poor-prognosis cancers