VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

Impact of drought stress on vitamin C and anthocyanin content in cultivated lettuces (Lactuca sativa L.) and wild relatives (Lactuca spp.).

Introduction: Lettuce production and quality could be seriously affected by the increasingly limited water resources. Methods: The effect of drought on the content of two antioxidant compounds, vitamin C and anthocyanins, in five cultivated lettuces and two wild relatives was assessed for 2 years. Results and discusion: In leaf samples, Lactuca wild species generally had a higher content of total vitamin C than the cultivated lettuces. In contrast, the commercial varieties usually contained more total anthocyanins than the wild species. Total vitamin C decreased with the drought stress in all accessions, commercial varieties, and lettuce wild relatives, with this tendency being consistent and reproducible across the 2 years. These differences were significant in the case of the green commercial varieties 'Winter Crop' (in 2020/2021) and 'Dolomiti G12' (in 2021/2022) and very significant in the red commercial variety 'Red Sails' (in 2020/2021). However, the only group in which the effect of drought was either significant or very significant in both years was the wild species, Lactuca homblei and Lactuca dregeana, and in the latter also in both tissues (leaf and stem) analyzed. Water stress resulted in an increase of the total anthocyanin content in the leaves from all the accessions, both red commercial varieties and wild relatives, in both years. The most significant enrichment and the only one being either significant or very significant in both years was observed in one of the wild relatives assayed (L. homblei). Stems (L. dregeana) contained more anthocyanins than leaves under control conditions, and it was exactly the opposite under drought. Changes in anthocyanins in the two tissues in response to drought stress were in opposite directions, increasing in leaves and decreasing in stems. This could suggest a translocation of anthocyanins as a first quick mechanism to cope with a severe lack of water. In conclusion, anthocyanins (unlike vitamin C) could play a role in the mechanisms deployed by the plant to tolerate drought stress. The wild species with a robust significant enrichment in anthocyanins as a response to drought (L. homblei) is a promising plant material to breed more resilient lettuces.

Pathogenic effects of Leu200Pro and Arg387His VRK1 protein variants on phosphorylation targets and H4K16 acetylation in distal hereditary motor neuropathy.

Rare recessive variants in the human VRK1 gene are associated with several motor neuron diseases (MND), such as amyotrophic lateral sclerosis, spinal muscular atrophy, or distal hereditary motor neuropathies (dHMN). A case with dHMN carrying two novel VRK1 gene variants, expressing Leu200Pro (L200P) and Arg387His (R387H) variant proteins, identified that these protein variants are functionally different. The Leu200Pro variant shares with several variants in the catalytic domain the loss of the kinase activity on different substrates, such as histones, p53, or coilin. However, the distal Arg387His variant and the distal Trp375* (W375X) chinese variant, both located at the end of the low complexity C-terminal region and proximal to the termination codon, retain their catalytic activity on some substrates, and mechanistically their functional impairment is different. The L200P variant, as well as most VRK1 pathogenic variants, impairs the phosphorylation of BAF and histone H4K16 acetylation, which are required for DNA attachment to the nuclear envelope and chromatin accessibility to DNA repair mechanisms, respectively. The R387H variant impairs phosphorylation of H2AX, an early step in different types of DNA damage responses. The functional variability of VRK1 protein variants and their different combinations are a likely contributor to the clinical phenotypic heterogeneity of motor neuron and neurological diseases associated with rare VRK1 pathogenic variants. KEY MESSAGES: VRK1 variants implicated in motor neuron diseases are functionally different. The L200P variant is kinase inactive, and the R387H variant is partially active. VRK1 variants alter H4K16 acetylation and loss of coilin and BAF phosphorylation. VRK1 variants alter Cajal bodies and DNA damage responses. VRK1 variant combination determines the neurological phenotype heterogeneity.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

A Novel Introgression Line Library Derived from a Wild Melon Gives Insights into the Genetics of Melon Domestication, Uncovering New Genetic Variability Useful for Breeding.

A collection of 30 melon introgression lines (ILs) was developed from the wild accession Ames 24297 (TRI) into 'Piel de Sapo' (PS) genetic background. Each IL carried an average of 1.4 introgressions from TRI, and the introgressions represented 91.4% of the TRI genome. Twenty-two ILs, representing 75% of the TRI genome, were evaluated in greenhouse (Algarrobo and Meliana) and field (Alcàsser) trials, mainly to study traits related to domestication syndrome such as fruit weight (FW) and flesh content (FFP), as well as other fruit quality traits as fruit shape (FS), flesh firmness (FF), soluble solid concentration (SSC), rind color and abscission layer. The IL collection showed an impressive variation in size-related traits, with FW ranging from 800 to 4100 g, reflecting the strong effect of the wild genome on these traits. Most of the ILs produced smaller fruits compared with PS; however, unexpectedly, the IL TRI05-2 produced bigger fruits, likely due to new epistatic interacions with the PS genetic background. In contrast, the genotypic effect for FS was smaller, and few QTLs with notable effects were detected. Interestingly, variability was also observed for FFP, FF and SSC, rind color and abscission layer formation. Genes in these introgressions are candidates for having been involved in melon domestication and diversification as well. These results confirm that the TRI IL collection is a very powerful tool for mapping traits of agronomic interest in melon, allowing the confirmation of previously reported QTLs and the identification of new ones to better understand the domestication process of this crop.

The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase.

BACKGROUND: Dynamic chromatin remodeling is associated with changes in the epigenetic pattern of histone acetylations and methylations required for processes based on dynamic chromatin remodeling and implicated in different nuclear functions. These histone epigenetic modifications need to be coordinated, a role that may be mediated by chromatin kinases such as VRK1, which phosphorylates histones H3 and H2A. METHODS: The effect of VRK1 depletion and VRK1 inhibitor, VRK-IN-1, on the acetylation and methylation of histone H3 in K4, K9 and K27 was determined under different conditions, arrested or proliferating cells, in A549 lung adenocarcinoma and U2OS osteosarcoma cells. RESULTS: Chromatin organization is determined by the phosphorylation pattern of histones mediated by different types of enzymes. We have studied how the VRK1 chromatin kinase can alter the epigenetic posttranslational modifications of histones by using siRNA, a specific inhibitor of this kinase (VRK-IN-1), and of histone acetyl and methyl transferases, as well as histone deacetylase and demethylase. Loss of VRK1 implicated a switch in the state of H3K9 posttranslational modifications. VRK1 depletion/inhibition causes a loss of H3K9 acetylation and facilitates its methylation. This effect is similar to that of the KAT inhibitor C646, and to KDM inhibitors as iadademstat (ORY-1001) or JMJD2 inhibitor. Alternatively, HDAC inhibitors (selisistat, panobinostat, vorinostat) and KMT inhibitors (tazemetostat, chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause increase of H3K9ac and a decrease of H3K9me3. VRK1 stably interacts with members of these four enzyme families. However, VRK1 can only play a role on these epigenetic modifications by indirect mechanisms in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1. CONCLUSIONS: The chromatin kinase VRK1 regulates the epigenetic patterns of histone H3 acetylation and methylation in lysines 4, 9 and 27. VRK1 is a master regulator of chromatin organization associated with its specific functions, such as transcription or DNA repair.

Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives.

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions.

Thorough Characterization of ETHQB3.5, a QTL Involved in Melon Fruit Climacteric Behavior and Aroma Volatile Composition.

The effect of the QTL involved in climacteric ripening ETHQB3.5 on the fruit VOC composition was studied using a set of Near-Isogenic Lines (NILs) containing overlapping introgressions from the Korean accession PI 16375 on the chromosome 3 in the climacteric 'Piel de Sapo' (PS) genetic background. ETHQB3.5 was mapped in an interval of 1.24 Mb that contained a NAC transcription factor. NIL fruits also showed differences in VOC composition belonging to acetate esters, non-acetate esters, and sulfur-derived families. Cosegregation of VOC composition (23 out of 48 total QTLs were mapped) and climacteric ripening was observed, suggesting a pleiotropic effect of ETHQB3.5. On the other hand, other VOCs (mainly alkanes, aldehydes, and ketones) showed a pattern of variation independent of ETHQB3.5 effects, indicating the presence of other genes controlling non-climacteric ripening VOCs. Network correlation analysis and hierarchical clustering found groups of highly correlated compounds and confirmed the involvement of the climacteric differences in compound classes and VOC differences. The modification of melon VOCs may be achieved with or without interfering with its physiological behavior, but it is likely that high relative concentrations of some type of ethylene-dependent esters could be achieved in climacteric cultivars.

Comparison of Accuracy of Color Spaces in Cell Features Classification in Images of Leukemia types ALL and MM / Comparación de Precisión de Espacios de Color en la Clasificación de Características de Células en Imágenes de Leucemia tipos ALL y MM

ABSTRACT This study presents a methodology for identifying the color space that provides the best performance in an image processing application. When measurements are performed without selecting the appropriate color model, the accuracy of the results is considerably altered. It is significant in computation, mainly when a diagnostic is based on stained cell microscopy images. This work shows how the proper selection of the color model provides better characterization in two types of cancer, acute lymphoid leukemia, and multiple myeloma. The methodology uses images from a public database. First, the nuclei are segmented, and then statistical moments are calculated for class identification. After, a principal component analysis is performed to reduce the extracted features and identify the most significant ones. At last, the predictive model is evaluated using the k-nearest neighbor algorithm and a confusion matrix. For the images used, the results showed that the CIE L*a*b color space best characterized the analyzed cancer types with an average accuracy of 95.52%. With an accuracy of 91.81%, RGB and CMY spaces followed. HSI and HSV spaces had an accuracy of 87.86% and 89.39%, respectively, and the worst performer was grayscale with an accuracy of 55.56%.

RESUMEN Este estudio presenta una metodología para identificar el espacio de color que proporciona el mejor rendimiento en una aplicación de procesamiento de imágenes. Cuando las mediciones se realizan sin seleccionar el modelo de color adecuado, la precisión de los resultados se altera considerablemente. Esto es significativo en el procesamiento, principalmente cuando el diagnóstico se basa en imágenes de microscopía de células teñidas. Este trabajo muestra cómo la selección adecuada del modelo de color proporciona una mejor caracterización en dos tipos de cáncer, la leucemia linfoide aguda y el mieloma múltiple. La metodología utiliza imágenes de una base de datos pública. Primero, se segmentan los núcleos y luego se calculan los momentos estadísticos para la identificación de clases. Posteriormente, se realiza un análisis de componentes principales para reducir las características extraídas e identificar las más significativas. Por último, el modelo predictivo se evalúa utilizando el algoritmo k-vecinos más cercanos y una matriz de confusión. Para las imágenes utilizadas, los resultados mostraron que el espacio de color CIE L*a*b caracterizó mejor los tipos de cáncer analizados con una precisión promedio del 95,52%. Con una precisión del 91,81%, siguieron los espacios RGB y CMY. Los espacios HSI y HSV tuvieron una precisión del 87,86% y el 89,39%, respectivamente, y el peor desempeño fue la escala de grises con una precisión del 55,56%.

Applications of Genomic Tools in Plant Breeding: Crop Biofortification.

Crop breeding has mainly been focused on increasing productivity, either directly or by decreasing the losses caused by biotic and abiotic stresses (that is, incorporating resistance to diseases and enhancing tolerance to adverse conditions, respectively). Quite the opposite, little attention has been paid to improve the nutritional value of crops. It has not been until recently that crop biofortification has become an objective within breeding programs, through either conventional methods or genetic engineering. There are many steps along this long path, from the initial evaluation of germplasm for the content of nutrients and health-promoting compounds to the development of biofortified varieties, with the available and future genomic tools assisting scientists and breeders in reaching their objectives as well as speeding up the process. This review offers a compendium of the genomic technologies used to explore and create biodiversity, to associate the traits of interest to the genome, and to transfer the genomic regions responsible for the desirable characteristics into potential new varieties. Finally, a glimpse of future perspectives and challenges in this emerging area is offered by taking the present scenario and the slow progress of the regulatory framework as the starting point.

Association between brain natriuretic peptide and cardiac dysfunction in hypertensive pregnancy disorders.

OBJECTIVE: The aim of this work is to identify the correlation between serum brain natriuretic peptide (BNP) and left ventricle (LV) systolic and diastolic function in hypertensive pregnancy disorders (HPD) through echocardiographic parameters. STUDY DESIGN: Eighty-seven pregnant patients were included, 23 with normotensive pregnancy (NP), 28 with gestational hypertension (GH) and 36 with preeclampsia (PE). Conventional 2D echocardiography was used to evaluate systolic and diastolic function such as E/a, E/e', LV mass index, LV ejection fraction, as well as left atria (LA) diameters, LA indexed volume, LV strain and LA strain (LAS). Brain natriuretic levels (BNP) blood levels were also determined. MAIN OUTCOME MEASURES: The serum levels of BNP were higher in patients with PE [median (interquartile range, IQR)] 189 (142-215) pg/ml and GH 105 (46-162) pg/ml compared to NP 23 (9-33) pg/ml, (p = 0.001). BNP levels had a negative correlation with LAS (Rho = -0.79, p < 0.001). Preeclampsia patients had lower LAS [median (IQR)] 22% (20%-24%) compared to the GH group 23% (20%-24%) and NP 35% (34% -35%), p = 0.001. Classification and Regression Trees multivariate analysis found patterns that define trends to form mutually excluding homogeneous groups such as: a) First parameter that separates 2 groups is septal e > 8.2 or < 8.2b), BNP serum levels above 89 pg/ml, and c) LAS increases the discriminatory performance to detect and define the diastolic dysfunction or not. CONCLUSIONS: At least one third of women with HPD had moderate diastolic dysfunction. The degree of diastolic dysfunction was negatively correlated with serum BNP levels and severity of HPD. LAS increase discriminatory performance to identify diastolic dysfunction in HPD.

Monosubstituted Coumarins Inhibit Epinephrine-induced Platelet Aggregation.

AIM: The aim of this study was to evaluate the in vitro effect of coumarin and 15 monosubstituted derivatives on the inhibition of human platelet aggregation induced by various proaggregatory agonists, particularly by epinephrine. BACKGROUND: The emergence of residual platelet reactivity during the use of conventional antiplatelet agents (acetylsalicylic acid and clopidogrel) is one of the main causes of double therapy´s therapeutic failure. Platelet adrenoceptors participate in residual platelet reactivity. Therefore, it is necessary to develop new antiplatelet agents that inhibit epinephrine-induced platelet aggregation as a new therapeutic strategy. Information on the antiplatelet activity of coumarins in inhibiting epinephrine-induced aggregation is limited. OBJECTIVE: The objective of this study was to establish the structure-activity relationship (SAR) of coumarin derivatives with hydroxy, methoxy, and acetoxy groups in different positions of the coumarin nucleus to identify the most active molecules. Moreover, this study aimed to use in silico studies to suggest potential drug targets to which the molecules bind to produce antiplatelet effects. METHODS: The platelet aggregation was performed using a Lumi-aggregometer; the inhibitory activity of 16 compounds were evaluated by inducing the aggregation of human platelets (250 × 103/µl) with epinephrine (10 µM), collagen (2 µg/ml) or ADP (10 µM). The aggregation of control platelets was considered 100% of the response for each pro-aggregatory agonist. RESULTS: Eleven molecules inhibited epinephrine-induced aggregation, with 3-acetoxycoumarin and 7-methoxycoumarin being the most active. Only coumarin inhibited collagen-induced platelet aggregation, but no molecule showed activity when using ADP as an inducer. CONCLUSIONS: In silico studies suggest that most active molecules might have antagonistic interactions in the α2 and ß2 adrenoceptors. The antiplatelet actions of these coumarins have the potential to reduce residual platelet reactivity and thus contribute to the development of future treatments for patients who do not respond adequately to conventional agents.

Consenso mexicano para el diagnóstico y tratamiento de la trombastenia de Glanzmann / Mexican consensus on the diagnosis and treatment of Glanzmann thrombasthenia

Resumen Las plaquetas tienen un papel central en diferentes escenarios fisiológicos, incluyendo la hemostasia; se unen unas con otras en la agregación plaquetaria, lo cual permite formar un coágulo plaquetario. Para que la agregación sea apropiada se requiere del complejo glicoproteico IIb/IIIa (GPIIb/IIIa) en la superficie plaquetaria. Toda alteración funcional plaquetaria, hereditaria o adquirida, impide la formación adecuada del coágulo y se manifiesta como hemorragia. Las enfermedades plaquetarias hereditarias son raras y, hasta recientemente, fueron ignoradas. Una de las más reconocidas y estudiadas es la trombastenia de Glanzmann (TG), entidad en la cual el número de plaquetas puede ser normal pero la función está alterada. Es un padecimiento autosómico y recesivo que causa hemorragia de diferente intensidad toda la vida y en la cual el problema radica en precisamente en la GPIIb/IIIa. Las hemorragias son típicamente mucocutáneas: equimosis, púrpura, epistaxis, gingivorragia; menos frecuentes son la hemorragia gastrointestinal, hemartrosis o en sistema nervioso central. La hiperpolimenorrea es común en las mujeres y llega a ser tan importante que amerita transfusiones en la menarca. La TG afecta a todos los grupos étnicos y su prevalencia varía entre 1/40,000 y 1/400,000. A pesar de esta información acerca de la TG en el mundo, hay pocas guías o recomendaciones basadas en la opinión de expertos y experiencias unicéntricas. En México la TG es rara y no se cuenta con una recomendación general para su diagnóstico y tratamiento. El objetivo de este documento fue establecer un consenso y hacer sugerencias generales para su diagnóstico y tratamiento.

Abstract Platelets have a central role in several physiological scenarios including hemostasis. Platelets bind each other during platelet aggregation allowing the proper formation of the clot; to be appropriate, platelet aggregation requires the glycoproteic complex IIb/IIIa (GPIIb/IIIa). Every platelet function abnormality both, congenital or acquired, impedes clot formation and favors bleeding episodes. Hereditary platelet abnormalities are rare and, until recently, they were almost ignored. Among these disorders, Glanzmann Thrombasthenia (GT) is a widely recognized abnormality in which platelet counts may be normal, but their function is affected. GT is an autosomal, recessive disease that causes life-long bleeding of different intensity. Main biochemical abnormality resides in GPIIb/IIIa. Bleeding is typically mucocutaneous: easy bruising, purpura, and nose and gum bleeds; less frequently are gastrointestinal bleeds, hemarthrosis, or intracranial. Menorrhagia and hyperpolymenorrhea are common findings in in women and may be the cause of anemia requiring blood transfusions at fertile age. GT affects all ethnic groups and its prevalence ranges between 1/40,000 to 1/400,000. Despite this worldwide information regarding GT, only a few guidelines and recommendations have been published, most of them based on expert opinions. In Mexico, GT is rare and there is not a general recommendation regarding its diagnosis and treatment. The aim of this document was to establish a consensus to suggest a general guideline for the diagnosis and treatment of GT in Mexico.

Nutritional value of commercial and traditional lettuce (Lactuca sativa L.) and wild relatives: Vitamin C and anthocyanin content.

Lettuce is the most consumed leafy vegetable though the most popular varieties have a low nutritional value. Our objective was to accurately quantify vitamin C and anthocyanins in wild relatives, and commercial and traditional varieties. Wild species and traditional varieties contained more total ascorbic acid (TAA) than commercial varieties (21% and 8%, respectively). In contrast, commercial varieties had significantly higher content of anthocyanins than traditional varieties and wild species (6 and 8 times more, respectively). TAA was significantly higher in green than in red lettuces (18%). TAA was also significantly higher in the leaves of two wild species than in stems. Cyanidin 3-O-(6'-O-malonylglucoside) was the most abundant anthocyanin (97%), present in most samples. The rankings of accessions by vitamin C and anthocyanin contents can be useful for consumers worried about the impacts of food on their wellbeing and for breeders aiming to improve lettuce by biofortification with health-promoting compounds.

Tyrame [N-(3-hydroxy-1:3:5(10)-estratrien-17ß-yl)-4-hydroxy-phenethylamine], antithrombotic aminoestrogen that decreases microvesicle formation.

BACKGROUND: Estrogens that are used as contraceptives or in replacement therapy are associated with an increase in the risk for developing thrombosis, mainly during the first year of treatment and in women with associated risk factors. OBJECTIVE: To synthesize, characterize and identify the anticoagulant, antiplatelet aggregation and microvesicle-reducing effect of the new aminoestrogen Tyrame. MATERIAL AND METHODS: CD1 strain mice were used, which had Tyrame (0, 1 and 2 mg/100 g) subcutaneously administered. At 24 h, a blood sample was obtained to determine whole-blood clotting time, microvesicles concentration and inhibitory effect on platelet aggregation. RESULTS: Blood clotting time increased up to 1.5 times in comparison with the control. Platelet aggregation inhibition had different magnitude depending on the agonist agent employed, and was complete with collagen. Both effects had a dose-dependent relationship. The microvesicles decreased up to six times with respect to the control. CONCLUSIONS: Tyrame reduces platelet aggregation and microvesicle formation, which emphasizes its potential therapeutic utility as an estrogen free of thrombotic effects.

ANTECEDENTES: Los estrógenos empleados como anticonceptivos o en la terapia de sustitución se asocian a un incremento en el riesgo de desarrollar trombosis, principalmente durante el primer año de tratamiento y en mujeres con factores de riesgo asociados. OBJETIVO: Sintetizar, caracterizar e identificar el efecto anticoagulante, antiagregante plaquetario y reductor de las microvesículas del nuevo aminoestrógeno tyrame. MATERIAL Y MÉTODOS: Se emplearon ratones cepa CD1 a los que se les administró por vía subcutánea tyrame (0, 1 y 2 mg/100 g). A las 24 h se tomó una muestra de sangre para determinar el tiempo de coagulación en sangre total, la concentración de microvesículas y el efecto inhibidor de la agregación plaquetaria. RESULTADOS: El tiempo de coagulación en sangre se incrementó hasta 1.5 veces con respecto al control. La inhibición de la agregación plaquetaria tuvo diferente magnitud dependiendo del agente agonista, siendo completa con colágena. Ambos efectos siguieron una relación dependiente de la dosis. Las microvesículas disminuyeron hasta seis veces con respecto al control. CONCLUSIONES: el tyrame disminuye la agregación plaquetaria y la formación de microvesículas, lo que acentúa su posible utilidad terapéutica como un estrógeno sin efectos trombóticos.

Parámetros de laboratorio de importancia en el manejo de pacientes con COVID-19 / Laboratory parameters of importance in the management of COVID-19 patients

Resumen El coronavirus 2 del síndrome respiratorio agudo grave (SARS-CoV-2) detona el padecimiento la enfermedad por coronavirus 2019 (COVID-19), poniendo en riesgo de muerte a la población vulnerable. El laboratorio clínico enfrenta un reto para el diagnóstico, seguimiento, pronóstico y evaluación de los tratamientos, que se ofrecen a los enfermos de COVID-19. Nuestro objetivo es mostrar al lector un resumen de los principales cambios en los parámetros que se estudian en los laboratorios clínicos. Material y métodos: Se hizo una búsqueda bibliográfica cruzando los términos COVID-19 y laboratorio clínico. Se analizaron las publicaciones relevantes por los miembros del Comité de Trombosis y Hemostasia-AMEH A.C. y se plasmaron las pruebas que a criterio de los participantes destacan por la relación entre la información que proporcionan respecto al seguimiento, pronóstico y evaluación al tratamiento. Resultados: Se recomienda solicitar una citometría hemática (recuento de células blancas, relación neutrófilo:linfocito), química sanguínea (transaminasas, bilirrubinas, albúmina, urea, creatinina, glucosa, lactato deshidrogenasa), pruebas de coagulación (tiempo de protrombina, tiempo de tromboplastina parcial activado, fibrinógeno y dímeros D) y pruebas especiales (proteína C reactiva, ferritina, procalcitonina, troponina).

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers the coronavirus disease 2019 (COVID-19), putting the vulnerable population at risk of death. The clinical laboratory faces a challenge for the diagnosis, monitoring, prognosis and evaluation of therapy with low molecular weight heparin. Our objective in this article is to offer a brief discuss of the main changes in the clinical parameters, studied on behalf of COVID-19 patients by a clinical laboratory. Material and methods: A bibliographic search was made crossing the terms COVID-19 and clinical laboratory. Relevant publications were analyzed by the members of the Committee for Thrombosis and Hemostasis-AMEH A.C. The relevant articles were discussed, and the clinical tests discussed in the article are those, that meet the criteria of providing information referring to the follow-up, prognosis and evaluation of treatment against the lower cost. Results: It is recommended to request a blood count (white cell count, neutrophyl/ lymphocytes ratio), clinical chemistry (transaminases, bilirubin, albumin, urea, creatinine, glucose, lactate dehydrogenase), coagulation tests (prothrombin time, activated partial thromboplastin time, fibrinogen, DD dimers) and special tests (C-reactive protein, ferritin, procalcitonin, troponins).

Fisiología de la hemostasia y su alteración por la coagulopatía en COVID-19 / Physiology of Hemostasis and its Alteration by Coagulopathy in COVID-19

Resumen La pandemia de la enfermedad COVID-19, ocasionada por el virus Sars-CoV-2, ha preocupado al personal de salud, entre otras cosas, por la alta incidencia de coagulopatía asociada a aumento en la mortalidad que se presenta en los pacientes. La coagulopatía es principalmente trombótica, inicialmente en pulmón y posteriormente sistémica, macro y microvascular, asociada al daño endotelial, inflamación, trampas extracelulares de neutrófilos (NETs), activación de macrófagos y tormenta de citocinas que perpetúan el círculo vicioso de trombosis e inflamación. Se ha reportado el aumento de factores protrombóticos en los pacientes: aumento del factor tisular, factor de Von Willebrand, fibrinógeno, factor VIII, entre otros y, además, la disminución de algunos anticoagulantes naturales como la proteína S y la antitrombina. Además, se menciona la insuficiencia de la fibrinólisis, asociada con el aumento del PAI-1 (inhibidor del activador tisular de plasminógeno). Durante la enfermedad, hay depósito de fibrina intraalveolar que también es degradada. Tanto la fibrinólisis del trombo, como la degradación de fibrina intraalveolar, hacen que aumenten los dímeros D y, por esta razón, este es uno de los mejores predictores de la severidad de la enfermedad COVID-19. En este artículo se revisa la fisiología de la hemostasia, la tromboinflamación secundaria a la infección por el virus Sars-Cov-2, la evidencia clínica y lo que se sabe de la fisiopatología de la coagulopatía en COVID-19, para tratar de entenderla desde la mirada de la ciencia básica.

Abstract COVID-19 global pandemic caused by Sars-CoV-2 virus, has worried to health care providers due to the high mortality rate related to coagulopathy in many patients. COVID-19 coagulopathy is mainly thrombotic, first locally in lungs but later on it becomes micro and macrovascular systemic coagulopathy. It has been associated to endothelial damage, inflammation, neutrophil-extracellular traps, monocyte and macrophage activation, cytokines storm that induce a vicious cycle of thrombosis and inflammation. The increased levels of prothrombotic factors as tissue factor, Von Willebrand factor, fibrinogen, VIII factor and the decreased levels of antithrombotic factos, such as: antithrombin and Protein S have been reported in COVID-19 patients. Insufficiency of fibrinolysis because of the increased levels of PAI-1 (plasminogen activator inhibitor 1) have been reported also. During this disease there are intraalveolar fibrin deposits that needs to be degraded. Fibrinolysis of thrombus and fibrin intraalveolar degradation are responsible for the high increase of D-dimers levels that are an important predictor of severity of the disease. In this report, the physiology of hemostasis, thromboinflamation secondary to Sars-CoV-2 infection are reviewed, as well as the clinical evidence and the physiopathology of COVID-19 coagulopathy from the basic sciences point of view.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.).

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized. Samples were collected to represent the entire plant, frozen at -80 °C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Correction to 'ALOX5, LPA, MMP9 and TPO gene polymorphisms increase atherothrombosis susceptibility in middle-aged Mexicans'.

[This corrects the article DOI: 10.1098/rsos.190775.].

ALOX5, LPA, MMP9 and TPO gene polymorphisms increase atherothrombosis susceptibility in middle-aged Mexicans.

Atherothrombosis is the cornerstone of cardiovascular diseases and the primary cause of death worldwide. Genetic contribution to disturbances in lipid metabolism, coagulation, inflammation and oxidative stress increase the susceptibility to its development and progression. Given its multifactorial nature, the multiloci studies have been proposed as potential predictors of susceptibility. A cross-sectional study was conducted to explore the contribution of nine genes involved in oxidative stress, inflammatory and thrombotic processes in 204 subjects with atherothrombosis matched by age and gender with a healthy group (n = 204). To evaluate the possibility of spurious associations owing to the Mexican population genetic heterogeneity as well as its ancestral origins, 300 unrelated mestizo individuals and 329 Native Americans were also included. ALOX5, LPA, MMP9 and TPO gene polymorphisms, as well as their multiallelic combinations, were twice to four times more frequent in those individuals with clinical manifestations of atherothrombosis than in the healthy group. Once adjusting for population stratification was done, these differences remained. Our results add further evidence on the contribution of ALOX5, LPA, MMP9 and TPO polymorphisms to atherothrombosis development in the middle-aged group, emphasizing the multiethnic studies in search of gene risk polymorphisms.