Transcription factor NAC1 activates expression of peptidase-encoding AtCEPs in roots to limit root hair growth.

Plant genomes encode a unique group of papain-type Cysteine EndoPeptidases (CysEPs) containing a KDEL endoplasmic reticulum (ER) retention signal (KDEL-CysEPs or CEPs). CEPs process the cell-wall scaffolding EXTENSIN (EXT) proteins that regulate de novo cell-wall formation and cell expansion. Since CEPs cleave EXTs and EXT-related proteins, acting as cell-wall-weakening agents, they may play a role in cell elongation. The Arabidopsis (Arabidopsis thaliana) genome encodes 3 CEPs (AtCPE1-AtCEP3). Here, we report that the genes encoding these 3 Arabidopsis CEPs are highly expressed in root-hair (RH) cell files. Single mutants have no evident abnormal RH phenotype, but atcep1-3 atcep3-2 and atcep1-3 atcep2-2 double mutants have longer RHs than wild-type (Wt) plants, suggesting that expression of AtCEPs in root trichoblasts restrains polar elongation of the RH. We provide evidence that the transcription factor NAC1 (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) activates AtCEPs expression in roots to limit RH growth. Chromatin immunoprecipitation indicates that NAC1 binds to the promoter of AtCEP1, AtCEP2, and, to a lower extent, AtCEP3 and may directly regulate their expression. Inducible NAC1 overexpression increases AtCEP1 and AtCEP2 transcript levels in roots and leads to reduced RH growth while the loss of function nac1-2 mutation reduces AtCEP1-AtCEP3 gene expression and enhances RH growth. Likewise, expression of a dominant chimeric NAC1-SRDX repressor construct leads to increased RH length. Finally, we show that RH cell walls in the atcep1-3 atcep3-2 double mutant have reduced levels of EXT deposition, suggesting that the defects in RH elongation are linked to alterations in EXT processing and accumulation. Our results support the involvement of AtCEPs in controlling RH polar growth through EXT processing and insolubilization at the cell wall.

Exploring the puzzle of Reactive Oxygen Species (ROS) acting on root hair cells.

Reactive oxygen species (ROS) are essential signaling molecules that enable cells to respond rapidly to a range of stimuli. The capacity of plants to recognize various stressors, incorporate a variety of environmental inputs, and initiate stress-response networks depends on ROS. Plants develop resilience and defensive systems as a result of these processes. Root hairs (RHs) are central components of the root biology since they increase the surface area of the root, anchor it in the soil, increase its ability to absorb water and nutrients, and foster interactions between microorganisms. In this review, we specifically focused on RHs cells and we highlighted the identification of ROS receptors, important new regulatory hubs that connect ROS production, transport, and signaling in the context of two hormonal pathways (auxin and ethylene) and under low temperature environmental input related to nutrients. As ROS plays a crucial role in regulating cell elongation rates, RHs are rapidly gaining traction as a very valuable single plant cell model for investigating ROS homeostasis and signaling. These promising findings might soon aid in the development of plants and roots that are more resilient to environmental stressors.

NMR Study on Laccase Polymerization of Kraft Lignin Using Different Enzymes Source.

The usage of laccases is a sustainable and environmentally friendly approach to modifying the Kraft lignin structure for use in certain applications. However, the inherent structure of Kraft lignin, as well as that resulting from laccase modification, still presents challenges for fundamental comprehension and successful lignin valorization. In this study, bacterial and fungal laccases were employed to modify eucalypt Kraft lignin. To evaluate the type and range of the chemical and structural changes of laccase-treated lignins, different NMR techniques, including solution 1H and 2D NMR (heteronuclear single quantum correlation (HSQC)), and solid-state 13C NMR, were applied. Size exclusion chromatography and infrared spectroscopy were also used. Interestingly, HSQC analysis showed substantial changes in the oxygenated aliphatic region of lignins, showing an almost complete absence of signals corresponding to side-chains due to laccase depolymerization. Simultaneously, a significant loss of aromatic signals was observed by HSQC and 1H NMR, which was attributed to a deprotonation of the lignin benzenic rings due to polymerization/condensation by laccase reactions. Then, condensed structures, such as α-5', 5-5', and 4-O-5', were detected by HSQC and 13C NMR, supporting the increment in molecular weight, as well as the phenolic content reduction determined in lignins.

The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth.

As sessile organisms, plants have evolved mechanisms to adapt to variable and rapidly fluctuating environmental conditions. Calcium (Ca2+) in plant cells is a versatile intracellular second messenger that is essential for stimulating short- and long-term responses to environmental stresses through changes in its concentration in the cytosol ([Ca2+]cyt). Increases in [Ca2+]cyt direct the strength and length of these stimuli. In order to terminate them, the cells must then remove the cytosolic Ca2+ against a concentration gradient, either taking it away from the cell or storing it in organelles such as the endoplasmic reticulum (ER) and/or vacuoles. Here, we review current knowledge about the biological roles of plant P-type Ca2+-ATPases as potential actors in the regulation of this cytosolic Ca2+ efflux, with a focus the IIA ER-type Ca2+-ATPases (ECAs) and the IIB autoinhibited Ca2+-ATPases (ACAs). While ECAs are analogous proteins to animal sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), ACAs are equivalent to animal plasma membrane-type ATPases (PMCAs). We examine their expression patterns in cells exhibiting polar growth and consider their appearance during the evolution of the plant lineage. Full details of the functions and coordination of ECAs and ACAs during plant growth and development have not yet been elucidated. Our current understanding of the regulation of fluctuations in Ca2+ gradients in the cytoplasm and organelles during growth is in its infancy, but recent technological advances in Ca2+ imaging are expected to shed light on this subject.

Induction of Extracellular Hydroxyl Radicals Production in the White-Rot Fungus Pleurotus eryngii for Dyes Degradation: An Advanced Bio-oxidation Process.

Among pollution remediation technologies, advanced oxidation processes (AOPs) are genuinely efficient since they are based on the production of strong, non-selective oxidants, mainly hydroxyl radicals (·OH), by a set of physicochemical methods. The biological counterparts of AOPs, which may be referred to as advanced bio-oxidation processes (ABOPs), have begun to be investigated since the mechanisms of induction of ·OH production in fungi are known. To contribute to the development of ABOPs, advanced oxidation of a wide number of dyes by the white-rot fungus Pleurotus eryngii, via a quinone redox cycling (QRC) process based on Fenton's reagent formation, has been described for the first time. The fungus was incubated with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe3+-oxalate, with and without Mn2+, leading to different ·OH production rates, around twice higher with Mn2+. Thanks to this process, the degradative capacity of the fungus increased, not only oxidising dyes it was not otherwise able to, but also increasing the decolorization rate of 20 dyes by more than 7 times in Mn2+ incubations. In terms of process efficacy, it is noteworthy that with Mn2+ the degradation of the dyes reached values of 90-100% in 2-4 h, which are like those described in some AOPs based on the Fenton reaction.

Enzyme-Catalyzed Polymerization of Kraft Lignin from Eucalyptus globulus: Comparison of Bacterial and Fungal Laccases Efficacy.

Kraft lignin, a side-stream from the pulp and paper industry, can be modified by laccases for the synthesis of high added-value products. This work aims to study different laccase sources, including a bacterial laccase from Streptomyces ipomoeae (SiLA) and a fungal laccase from Myceliophthora thermophila (MtL), for kraft lignin polymerization. To study the influence of some variables in these processes, a central composite design (CCD) with two continuous variables (enzyme concentration and reaction time) and three levels for each variable was used. The prediction of the behavior of the output variables (phenolic content and molecular weight of lignins) were modelled by means of response surface methodology (RSM). Moreover, characterization of lignins was performed by Fourier-transform infrared (FTIR) spectroscopy and different nuclear magnetic resonance (NMR) spectroscopy techniques. In addition, antioxidant activity was also analyzed. Results showed that lignin polymerization (referring to polymerization as lower phenolic content and higher molecular weight) occurred by the action of both laccases. The enzyme concentration was the most influential variable in the lignin polymerization reaction within the range studied for SiLA laccase, while the most influential variable for MtL laccase was the reaction time. FTIR and NMR characterization analysis corroborated lignin polymerization results obtained from the RSM.

Peripheral double negative T: A look at senescent Cubans.

INTRODUCTION: Age-related changes in the immune system are called immunosenescence. Within the T lymphocytes is the subpopulation of double negative (DNT) peripheral lymphocytes that are immunomodulators of the immune response, based on their ability to suppress the functions of simple positive T cells and their cytotoxicity for tumor cells and those infected by viruses. OBJECTIVE: To determine the frequency of peripheral DNT lymphocytes in older Cuban adults. METHODS: A cross-sectional study was carried out in 30 older adults, residents in Cuba. DNT lymphocytes in peripheral blood were quantified by flow cytometry. A Beckman Coulter Gallios flow cytometer was used for data reading and analysis. Percentage values mean and standard deviation were used. The Chi-square was used to relate the percentage values of DNT and comorbidities. It was considered statistically significant if p ≤ 0.05. RESULTS: There was a predominance of women who represented 70 %. No older adult with low values of DNT lymphocytes was reported. Women with high percentage and absolute values of DNT lymphocytes prevailed in relation to men. In the group ≥80 years, high values in % and absolute values of DNT lymphocytes predominated. The high percentage values of DNT cells were mainly related to cardiovascular disease, and predominated in the elderly of ≥80 years old; who presented respiratory and skin infections, fundamentally. The percentage normal value in the group < 80 years was significant (p = 0.0198). The Chi-square value was 0,5995. CONCLUSIONS: Most older adults who exhibited high percentage and absolute values of DNT lymphocytes, or a tendency to them, had some associated comorbidity, an idea that suggests that DNT cells participate in immune surveillance, defense and homeostasis based on their double identity, that is, its pathogenic or immunosuppressive phenotype according to the specific immunological microenvironment.

Immunophenotypic characterization of B1a lymphocytes in Cuban older adults.

INTRODUCTION: B1a lymphocytes are characterized by having a high capacity for self-renewal and production of natural antibodies, in a T-independent manner. There are differences in both the number and composition of mature B lymphocytes throughout life, due to the phenomenon of immunosenescence. OBJECTIVE: To characterize the immunophenotype of B1a lymphocytes in older Cuban adults with a simplified CD19CD20CD5 panel. METHODS: A cross-sectional study was conducted in 30 institutionalized Cuban older adults. Characterization of CD19 + CD5+, CD20 + CD5+, and CD19 + CD20+ B lymphocytes in peripheral blood was performed by flow cytometry. For the reading and analysis of the biological samples, a flow cytometer, Beckman Coulter, Gallios, was used. A Microsoft Excel database was created with the information obtained. The sample was divided by sex and age. The data were processed using the statistical program GraphPadPrism version 6.00 using the percentage values and the median for the sample description. RESULTS: The elderly aged 80 years and over showed a decrease in the absolute count of CD19 + CD20+ B lymphocytes, as well as in the percentage and absolute count of CD19 + CD5+ B lymphocytes. On the other hand, they presented higher absolute counts of CD20 + CD5+ lymphocytes in relation to those of the <80-year-old group. Women showed higher absolute counts of CD19 + CD5+, CD20 + CD5+, and CD19 + CD20+ B lymphocyte populations. CONCLUSIONS: The immunophenotypic characterization of B1a lymphocytes in older Cuban adults is similar to that reported by other researchers. Both age and sex influence the absolute count of these cells, being higher in women under 80 years of age.

Biomarkers of sequela in adult patients convalescing from COVID-19.

Different biomarkers for SARS-CoV-2 have been linked to detection, diagnosis, treatment, disease progression, and development of new drugs and vaccines. The objective of this research was to evaluate various hematological, biochemicals, immunological, radiological and spirometric parameters in 20 adult patients convalescing from COVID-19 and their possible relationship with the clinical course of the disease. The frequencies of categorical variables were compared using the chi-square and Fisher's exact test. The levels of statistical significance were denoted in each figure legend. Two-dimensional clustering analysis was performed using MeV software from TIGR. The tests with P value of ≤ 0.05 were considered statistically significant. Most of the patients studied presented alterations in dissimilar laboratory, radiological and spirometric parameters, which were related to the clinical evolution of the disease. The results obtained show that certain hematological, biochemical, immunological and radiological parameters can be considered as biomarkers of sequela in adult COVID-19 patients, which allows their stratification, according to the degree of involvement or sequela, into three groups: I (mild degree of involvement or sequela), without lung lesions on computerized axial tomography (CT scan) and high values of IgG, C3 and hemoglobin, II (moderate degree of involvement or sequel), without lung lesions on CT scan, characterized by high levels of CD3+/CD4+ T lymphocytes and the rest of the variables with low values and III (severe degree of involvement or sequela), with lung lesions on CT scan and high values of erythrocyte sedimentation rate, monocytes and neutrophils, associated with lymphopenia and decreased concentrations of IgG and C3.

Assessment of Sustainability of Bio Treated Lignocellulose-Based Oleogels.

The development of biological strategies to obtain new high-added value biopolymers from lignocellulosic biomass is a current challenge for scientific community. This study evaluates the biodegradability and ecotoxicity of new formulated oleogels obtained from fermented agricultural residues with Streptomyces, previously reported to show improved rheological and tribological characteristics compared to commercial mineral lubricants. Both new oleogels exhibited higher biodegradation rates than the commercial grease. Classical ecotoxicological bioassays using eukaryotic organisms (Lactuca sativa, Caenorhabditis elegans) showed that the toxic impact of the produced bio-lubricants was almost negligible and comparable to the commercial grease for the target organisms. In addition, high throughput molecular techniques using emerging next-generation DNA-sequencing technologies (NGS) were applied to study the structural changes of lubricant-exposed microbial populations of a standard soil. Results obtained showed that disposal of biomass-based lubricants in the soil environment did not substantially modify the structure and phylogenetic composition of the microbiome. These findings point out the feasibility and sustainability, in terms of biodegradability and eco-safety, of the new bio-lubricants in comparison with commercial mineral greases. This technology entails a promising biological strategy to replace fossil and non-renewable raw materials as well as to obtain useful biopolymers from agricultural residues with potential for large-scale applications.

Immunophenotypic characterization of double positive T lymphocytes in Cuban older adults.

INTRODUCTION: Immunosenescence is the functional deterioration of the immune system during natural aging that mainly affects T lymphocytes, within which is the subpopulation of peripheral double positive T lymphocytes that are immunomodulators of the immune response. They are increased in older adults and play a fundamental role in the immunopathogenesis of autoimmune diseases, viral infections and neoplasms. OBJECTIVE: To characterize the immunophenotype of peripheral double positive T lymphocytes in Cuban older adults. METHODS: A cross-sectional study was carried out in 30 older adults residing in Cuba. Peripheral blood double positive T lymphocytes were quantified by flow cytometry. The reading was done on a Beckman Coulter Gallios flow cytometer. A Beckman Coulter Gallios flow cytometer was used for data analysis. Percentage values, median and standard deviation were used. RESULTS: There was a predominance of women who represented 70%. No older adults with low values of double positive T lymphocytes were reported. Women with high percentage and absolute values of DPT lymphocytes predominated in relation to men. Among the high percentage values and the absolute value of DPT lymphocytes, the group ≥80 years prevailed. CONCLUSIONS: Most of the older adults who exhibited high percentage and absolute values of DPT lymphocytes had some associated comorbidity, an idea that suggests that CD4 + CD8+ T cells seem to play an important role in peripheral sites as powerful immunosuppressants or as cells with high cytotoxic potential.

Effect of Biomodulina-T® and VA-MENGOC-BC® on lymphocyte subpopulations in older adults.

INTRODUCTION: The elderly population suffers from the natural process called immunosenescence, which may be related to the high mortality rates it has against the SARS-CoV2 virus, which is why therapies that improve the immune status are required. The combined treatment of the VA-MENGOC-BC® (V-BC) vaccine and the Biomodulina T® (BT) drug could achieve this purpose. This treatment could immunomodulate both the innate and adaptive branches of the immune system simultaneously. OBJECTIVE: To determine the effect of BT and V-BC on the immunomodulation of lymphocyte subpopulations in older adults. METHODS: Our study was carried out in 30 apparently healthy Cuban adults over 65 years of age. The study included three groups of 10 subjects per treatment: a combination of both and the monotherapies. Before and 7 days after treatment, 2 mL of peripheral blood was drawn from each subject. Multiparametric flow cytometry was used to identify lymphocyte subpopulations. For the comparison between the groups, point estimates and the confidence intervals of the Odds Ratio were made. RESULTS: We found that subpopulations of B lymphocytes and natural cytotoxic T (NKT) cells increased only with the administration of BT. Additionally, combination treatments and V-BC did not generate statistically significant immunomodulatory changes in any of the studied lymphocyte subpopulations. CONCLUSIONS: BT presented an immunoenhancing effect on the B and NKT lymphocyte subpopulations of older adults. The three-dose treatment scheme a novel and specific treatment strategy for this formulation. We also were verified that the combined application of V-BC and BT did not have the expected benefits. All these findings suggest that BT administration is a promising approach for immune restoration and to offering protection in elderly patients against COVID-19.

Calcium-regulated chloride channel anoctamin-1 is present in the suprachiasmatic nuclei of rats.

Calcium-regulated chloride channel (CaCC) anoctamin-1 has been recently identified in neurons. In neurons, which express the Na-K-2Cl cotransporter, activation of CaCCs increases firing frequency, by reversion between the Cl equilibrium potential and the membrane resting potential, leading to membrane depolarization by Cl extrusion from the cell. Although there are no reports of CaCCs present in the suprachiasmatic nuclei (SCN), the fact that Na-K-2Cl cotransporter is present in SCN neurons, where it has been shown to be involved in the excitatory effects of γ-aminobutyric acid, together with the increase of neuronal firing rate induced by release of intracellular Ca after administration of 100 nM ryanodine, leads us to determine whether CaCCs are present in the SCN. Immunohistochemistry and western blots show the expression of the CaCCs anoctamin-1 protein. Quantitative PCR demonstrated the expression of anoctamin-1 mRNA in the SCN. These results clearly indicate the presence of CaCC in SCN of rats.

Longitudinal gradient coils with enhanced radial uniformity in restricted diameter: Single-current and multiple-current approaches.

An important requirement for a gradient coil is that the uniformity of the generated magnetic field gradient should be maximal within the active volume of the coil. For a cylindrical geometry, the radial uniformity of the gradient turns critic, particularly in cases where the gradient-unit has to be designed to fit into the inner bore of a compact magnet of reduced dimensions, like those typically used in fast-field-cycling NMR. In this paper we present two practical solutions aimed to fulfill this requirement. We propose a matrix-inversion optimization algorithm based on the Biot-Savart law, that using a proper cost function, allows maximizing the uniformity of the gradient and power efficiency. The used methodology and the simulation code were validated in a single-current design, by comparing the computer simulated field map with the experimental data measured in a real prototype. After comparing the obtained results with the target field approach, a multiple-element coil driven by independent current sources is discussed, and a real prototype evaluated. Opposed equispaced independent windings are connected in pairs conforming an arrangement of independent anti-Helmholtz units. This last coil seizes 80% of its radial dimension with a gradient uniformity better than 5%. The design also provides an adaptable region of uniformity along with adjustable coil efficiency.

Measurement of the bending elastic modulus in unilamellar vesicles membranes by fast field cycling NMR relaxometry.

The elastic properties of lipid membranes can be conveniently characterized through the bending elastic modulus κ. Elasticity directly affects the deformability of a membrane, morphological and shape transitions, fusion, lipid-protein interactions, etc. It is also a critical property for the formulation of ultradeformable liposomes, and of interest for the design of theranostic liposomes for efficient drug delivery systems and/or different imaging contrast agents. Measurements of κ in liposome membranes have been made using the fast field cycling nuclear magnetic relaxometry technique. We analyze the capability of the technique to provide a consistent value of the measured quantity under certain limiting conditions. Relaxation dispersions were measured acquiring a minimal quantity of points, within a reduced Larmor frequency range and, under inferior experimental conditions (in the presence of magnetic field in-homogeneity and lower power supply stability). A simplified model is discussed, showing practical advantages when fitting the data within the reduced frequency range. Experiments are contrasted with standard measurements performed in a state-of-the-art relaxometer. The methodology was tested in samples of 1,2-dimyristoyl-sn-glycero-3-phosphocholine with different percentiles of cholesterol. We observe a tendency to a decrease in κ with increasing temperature, and a tendency to increase with the cholesterol percentile.

Estudio inmunológico en paciente con síndrome de Behcet: reporte de un caso / immunological study in a patient with Behcet Syndrome: a case report

Introducción: El síndrome de Behcet, o enfermedad de Behcet, es un proceso autoinflamatorio crónico, poco frecuente, de etiología desconocida. Es una vasculitis que afecta arterias y venas de todos los calibres, con alteración de la función endotelial, que se expresa clínicamente con lesiones orgánicas variadas. En su fisiopatogenia intervienen factores genéticos, microbianos e inmunológicos. Los síntomas más comunes son las úlceras orales y genitales, inflamaciones oculares (uveítis, retinitis e iritis), lesiones de piel y artritis. Objetivo: Evaluar diversos marcadores de la respuesta inmune en paciente con síndrome de Behcet. Presentación del caso: Paciente masculino. 39 años de edad, con diagnóstico clínico de enfermedad de Behcet con reactantes de fase aguda y marcadores serológicos de autoinmunidad negativa. Las subpoblaciones linfocitarias están dentro de los valores referenciales, sin evidencias de activación linfocitaria. La presencia de una doble población de linfocitos B y los antecedentes familiares, sugieren la existencia de una población de linfocitos B de autoreconocimiento y la posible presencia de factores genéticos, respectivamente. El paciente respondió favorablemente a la terapia con esteroides. Conclusiones: El estudio apoya el criterio de que, en condiciones basales, no se detectan marcadores humorales de autoinmunidad, alteraciones de los valores de las subpoblaciones linfocitarias, ni evidencias de activación linfocitaria, pero no se puede excluir la presencia de una población de linfocitos B de autoreconocimiento(AU)

Introduction: Behcet's syndrome, also known as Behcet's disease is a chronic autoinflammatory process of low frequency and unknown etiology. It's an all sizes arteries and veins affecting vasculitis that causes an alteration of endothelial function and is expressed clinically by organ damage at various levels. Its pathogenesis involves genetic, microbial and immunological factors. The most common symptoms are oral and genital ulcers, eye inflammation (uveitis, iritis and retinitis), skin lesions and arthritis. Objective: to evaluate several inmunological markers in a patient with Behcet syndrome. Case presentation: 39 years old masculine patientwith clinical diagnosis of Behcet disease with negative acute phase reactants and serological authoinmunity markers and lymphocyte populations within referential range, without evidences of lymphocyte activation. The presence of a double B lymphocyte population and familial background, suggest the presence of a self recognitionB lymphocyte population and the probable presence of genetic factors, respectively. There was a good response to steroids treatment. Conclusions: The study supports the idea that at baseline, not humoral autoimmunity markers, changes in the values of lymphocyte subpopulations, and evidence of lymphocyte activation is detected, but can not exclude the presence of a population of B lymphocytes self-recognition(AU)

Diagnóstico, clasificación y tratamiento de la leucemia aguda de linaje ambiguo / Diagnosis, classification and treatment of acute leukemia of ambiguous lineage

Introducción: Las leucemias agudas de linaje ambiguo constituyen un grupo heterogéneo de leucemias agudas que no muestran una evidencia clara de diferenciación a lo largo del linaje celular. El diagnóstico definitivo se realiza exclusivamente por las características inmunofenotípicas, con el uso de la citometría de flujo multiparamétrica. Objetivo: Actualizar los criterios diagnósticos, la clasificación, el manejo clínico y terapéutico de las leucemias agudas de linaje ambiguo. Métodos: Se realizó una búsqueda de información científica relacionada con el tema en libros de textos de Hematología Clínica y en artículos publicados a través de PUBMED en los últimos 10 años. Se hizo un análisis y resumen de la bibliografía revisada. Análisis y síntesis de la información: Las características morfológicas de las células leucémicas procedentes de los pacientes con leucemias agudas de linaje ambiguo no muestran uniformidad. Pueden encontrarse de forma simultánea blastos de diferentes tamaños y estados de maduración. Estos pueden presentar apariencia linfoide, mieloide, indiferenciada o mixta. Conclusión: Las leucemias agudas de linaje ambiguo constituyen un subgrupo de alto riesgo, con un pronóstico desfavorable y una pobre supervivencia global de los enfermos. Su reporte oportuno es indispensable para aunar los criterios de la toma de decisiones terapéuticas en este grupo de pacientes(AU)

Introduction: Acute leukemias of ambiguous lineage constitute a heterogeneous group of acute leukemias that do not show clear evidence of differentiation along cell lineage. The definitive diagnosis is made exclusively based on immunophenotypic characteristics, with the use of multiparametric flow cytometry. Objective: To update the diagnostic criteria, classification, as well as the clinical and therapeutic management of acute leukemias of ambiguous lineage. Methods: A search for scientific information related to the subject was carried out in clinical hematology textbooks and in articles published through PUBMED in the last ten years. An analysis and summary of the revised bibliography was carried out. Information analysis and synthesis: The morphological characteristics of the leukemic cells from patients with acute leukemia of ambiguous lineage do not show uniformity. Blasts of different sizes and stages of maturation can be found simultaneously. These may appear as lymphoid, myeloid, undifferentiated, or mixed. Conclusion: Acute leukemias of ambiguous lineage constitute a high-risk subgroup, with unfavorable prognosis and poor overall patient survival. Its timely report is essential to gather criteria for making therapeutic decisions in this group of patients(AU)

Avances en el diagnóstico de la enfermedad granulomatosa crónica: primer estudio familiar en Cuba / Advances in the diagnosis of chronic granulomatous disease: the first family study in Cuba

Introducción: La enfermedad granulomatosa crónica es una inmunodeficiencia primaria causada por mutaciones en la enzima NADPH oxidasa. Esta compromete la producción de especies reactivas del oxígeno, que son importantes contra patógenos. La prueba de la oxidación de la dihidrorodamina es un método eficaz para diagnosticar la enfermedad. Objetivo: Demostrar la utilidad de la prueba de la oxidación de la dihidrorodamina y del patrón de herencia en la confirmación del diagnóstico de la enfermedad granulomatosa crónica de un paciente. Métodos: Estudio de caso de una familia con diagnóstico de enfermedad granulomatosa crónica. Se tomó muestra de sangre periférica para citometría de flujo a tres individuos. Se realizó la prueba de la oxidación de la dihidrorodamina bajo estímulo con acetato de forbolmiristato y se evaluaron las subpoblaciones linfocitarias. Las muestras se leyeron en un citómetro GALLIOS, Beckman Coulter. Los datos obtenidos se analizaron en el programa informático Kaluza. Resultados: El paciente masculino tuvo un valor de oxidación de la dihidrorodamina positiva de 0,87 por ciento, que confirmó un patrón de herencia ligado al cromosoma X; mientras que la madre y hermana gemela portadoras tuvieron valores de 46,76 por ciento y 37,32 por ciento, respectivamente. Se encontraron alteraciones en las subpoblaciones linfocitarias. Conclusiones: La prueba de la oxidación de la dihidrorodamina es un método muy efectivo, rápido y sencillo que confirma el diagnóstico de la enfermedad granulomatosa crónica y determina el patrón de herencia y fenotipo de la enfermedad. Además, permite identificar a las mujeres portadoras según la distribución de los neutrófilos normales y los que tienen el gen CYBB mutado(AU)

Introduction: Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the NADPH oxidase enzymes. This compromises the production of oxygen reactive species, which are important against pathogens. The dihydrorhodamine oxidation test is an effective method for diagnosing the disease. Objective: To demonstrate the usefulness of the dihydrorhodamine oxidation test and the inheritance pattern in confirming the diagnosis of chronic granulomatous disease in a patient. Methods: A case study of a family with a diagnosis of chronic granulomatous disease. A peripheral blood sample was taken from three individuals and by flow cytometry. The dihydrorhodamine oxidation test was performed under stimulation with phorbolmyristate acetate, and lymphocyte subpopulations were evaluated. The samples were read on a GALLIOS, Beckman Coulter cytometer. The data obtained were analyzed using the computer program Kaluza. Results: The male patient had a positive dihydrorhodamine oxidation value of 0.87 percent, which confirmed an inheritance pattern linked to the X chromosome; while the carrier mother and twin sister had values 8203;8203;of 46.76 percent and 37.32 percent, respectively. Alterations were found in the lymphocyte subpopulations. Conclusions: The dihydrorhodamine oxidation test is a very effective, fast and simple method that confirms the diagnosis of chronic granulomatous disease and determines the inheritance pattern and phenotype of the disease. In addition, it allows the identification of female carriers according to the distribution of normal neutrophils and those with the CYBB mutation(AU)

[Echocardiographic screening vs. symptomatic diagnosis for patent ductus arteriosus in preterms]. / Pesquisa o diagnóstico sintomático por ecocardiografía en la persistencia del conducto arterioso en prematuros.

BACKGROUND: The persistence of ductus arteriosus, the aim of this study is to evaluate the possible benefit in the treatment for ductus closure when an early (less than 72 hours of life) echocardiographic screening is done versus when the echocardiographic diagnosis is realized only in present of associated symptoms. METHODS: Preterm without malformation followed by two strategies: patent ductus arteriosus (PDA) screening or echocardiographic study on suspected PDA for symptoms (control group). We analyzed the ductus characteristics, the presence of pulmonary hypertension and the treatments for their closure. We analyze the result in relation of the premature age as late preterm (34 to 36 gestational age weeks), moderate (30 to 33) and extreme (< 30). RESULTS: There was no difference in the proportion of newborns diagnosed with PDA among the strategies (screening 18.6 % [101/543] vs 18.1 % [55/304], p = 0.92). In the control group, 53 % were diagnosed after 72 hours of life. There were no differences in relation of the ductus characteristic among the groups. The closure treatment were similar in the two strategies among the moderate and late preterm, but was more aggressive in the control group in the extreme preterms. CONCLUSIONS: The echocardiographic screening for PDA in extreme preterm newborns reduces the time to detect it, and allow to give less aggressive treatment. We do not recommend their use in moderate o late preterm newborn.

Introducción: el objetivo es evaluar el posible beneficio en el tratamiento en el cierre de la persistencia del conducto arterioso cuando se realiza una pesquisa (< 72 horas de vida) por ecocardiograma, que cuando se realiza solo ante la presencia de síntomas asociados. Métodos: niños pretérmino sin malformaciones en dos estrategias diagnósticas: por pesquisa (asintomáticos) y por sintomatología asociada a la persistencia del conducto arterioso (PCA). Se analizaron las características del conducto, la presencia de hipertensión pulmonar y el tratamiento recibido. Se compararon los resultados entre: tardíos (34 a 36 semanas de gestación [SEG]), moderados (30 a 33 SEG) y extremos (< 30 SEG). Resultados: no hubo diferencias en las proporciones de diagnósticos de PCA por estrategias (pesquisa 18.6 % [101/543] contra 18.1 % [55/304], p = 0.92). Sin pesquisa, el 53 % se diagnosticó después de las 72 horas de vida. No hubo diferencias en las características del conducto entre los grupo. El tratamiento recibido no difirió entre las estrategias en los prematuros moderados y tardíos, pero sí fue más agresivo en los extremos. Conclusiones: la pesquisa de una PCA por ecocardiografía en los infantes pretérmino extremos acorta el tiempo de detección y permite un tratamiento menos agresivo. No lo recomendamos en pretérminos moderados y tardíos.

Diagnóstico por citometría de flujo de la hemoglobinuria paroxística nocturna / Diagnosis of paroxysmal nocturnal hemoglobinuria by flow cytometry

Introducción: La hemoglobinuria paroxística (HPN) nocturna es una enfermedad clonal, adquirida y no maligna de la célula madre hematopoyética. En este padecimiento se encuentra afectado el anclaje a la membrana celular de moléculas como el CD55 y CD59, fundamentales en la regulación de la lisis mediada por el complemento. Por su elevada especificidad y sensibilidad, la citometría de flujo multiparamétrica (CFM) es el método de elección para el diagnóstico de esta enfermedad. Objetivo: Establecer un algoritmo diagnóstico de la HPN por CMF. Métodos: Se analizó una muestra de sangre periférica para CFM de un paciente con sospecha de HPN. El inmunofenotipaje celular se realizó con un panel de anticuerpos monoclonales dirigidos contra los antígenos que se expresan en la membrana citoplasmática mediante su anclaje al glicosilfosfatidilinositol. Las muestras se leyeron en un citómetro GALLIOS, Beckman Coulter y los datos obtenidos se analizaron con el empleo del programa informático Kaluza. Resultados: Se identificaron cuatro clones HPN. En los granulocitos se observó un clon HPN de aproximadamente 90 por ciento, con deficiencia en la expresión de CD16, CD24, CD55 y CD59. En los monocitos se observaron dos clones: (1) clon CD14_CD59_ y (2) clon CD14_CD59+ con tamaños clonales de 59,77 por ciento y 19,45 por ciento, respectivamente. En los eritrocitos se identificó un clon de 19,98 por ciento y de determinó el grado de afectación. Conclusiones: El algoritmo de análisis propuesto permite identificar las poblaciones celulares con clones HPN. Además, dichos clones pueden ser cuantificados en cuanto a tamaño clonal y expresividad de los antígenos dependientes de anclaje a glicosilfosfatidilinositol. Con la CFM se logra determinar con elevada sensibilidad el grado de afectación de los eritrocitos en la expresión de CD59 como medida directa de la susceptibilidad que experimentan a la lisis por el complemento(AU)

Introduction: The paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, acquired disease and not malignant hematopoietic stem cell. In this condition, the anchor to the cell membrane of molecules such as the CD55 and CD59 is affected, This antigens are fundamental in the regulation of the complement-mediated lysis. By its high specificity and sensitivity multiparametric flow cytometry (MFC) is the goal standard for the diagnosis of this disease. Objective: To establish a diagnosis of PNH by MFC algorithm. Methods: A sample of peripheral blood of a patient with suspicion of PNH was analyzed by MFC. The cell immunophenotyping was carried out using a panel of monoclonal antibodies directed against antigens that are expressed in the cytoplasmic membrane through its the glycosylphosphatidylinositol anchor. The samples were read in a Cytometer GALLIOS, Beckman Coulter and the data obtained were analyzed with the use of the Kaluza software. Results: We identified four clones HPN. A HPN clone of approximately 90 percent, was observed in granulocytes with deficiency in the expression of CD16, CD24, CD55, CD59. In the monocytes were two clones: (1) CD14-CD59- clone and (2) CD14-CD59 + clone, with size clone of 59.77 percent and 19.45 percent, respectively. A clone of 19.98 percent was identified in erythrocytes and determined the degree of involvement of the same. Conclusions: The proposed analysis algorithm allows to identify cellular populations with clones PNH. In addition, these clones can be quantified in terms of size clonal and expressiveness of anchor to glycosylphosphatidylinositol antigen dependent. With the MFC is achieved with high sensitivity to determine the degree of involvement of the erythrocytes in the expression of CD59 as a direct measure of susceptibility undergoing lysis by complement(AU)