La miocardiopatía hipertrófica induce cambios en la expresión de la triptófano-5-hidroxilasa, el transportador de serotonina y receptores serotoninérgicos / Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions

Resumen Introducción: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. Objetivo: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). Métodos: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. Resultados: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). Conclusiones: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Abstract Introduction: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. Objective: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). Methods: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. Results: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). Conclusions: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

Diabetes mellitus causes changes in the activity and expression of brain tryptophan-5-hydroxylases and in the number of serotonergic neurons, which do not return to normal with insulin treatment.

INTRODUCTION: Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. OBJECTIVES: To determine whether DM-induced changes in brain TPH1 or TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. METHODS: Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expression of TPH1 and TPH2 by West-ern blot, and the number of serotonergic neurons by immunohistochemistry. RESULTS: In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine, and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hy-droxytryptamine, TPH expression, or the number of serotonergic neurons. CONCLUSIONS: DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

INTRODUCCIÓN: La diabetes mellitus (DM) inhibe la biosíntesis de serotonina cerebral mediante cambios en la actividad y expresión de la triptófano-5-hidroxilasa (TPH). OBJETIVOS: Determinar si los cambios en la expresión de TPH1 o TPH2 cerebral y en el número de neuronas serotoninérgicas causados por la DM retornan a la normalidad en las ratas con diabetes tratadas con insulina. MÉTODOS: Ratas con diabetes inducida con estreptozotocina se dividieron en dos grupos: uno tratado con insulina y otro sin tratamiento. En el día 14, se obtuvieron tallos cerebrales para cuantificar niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH. La expresión de TPH1 y TPH2 fue mediante Western blot y el número de neuronas serotoninérgicas por inmu­nohistoquímica. RESULTADOS: En las ratas con diabetes se confirmó disminución de los niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH, así como una menor expresión de TPH1 y 2 y un menor número de neuronas serotoninérgicas. Cuando las ratas diabéticas fueron tratadas con insulina, el L-triptófano regreso a la normalidad, no así la 5-hidroxitriptamina, la expresión de TPH y el número de neuronas serotoninérgicas. CONCLUSIONES: La DM inhibe crónicamente la síntesis de 5-hidroxitriptamina cerebral mediante modificaciones en la expresión de TPH1 y TPH2 y disminución de las neuronas seroto­ninérgicas, que persisten a pesar del tratamiento con insulina.

Cambios en la actividad y expresión de las triptófano-5-hidroxilasas cerebrales y en el número de neuronas serotoninérgicas por la diabetes mellitus, que no retornan a la normalidad con la insulina / Diabetes mellitus causes changes in brain tryptophan-5-hydroxylases activity and expression and in the number of serotonergic neurons, which do not return to normal with insulin treatment

Resumen Introducción: La diabetes mellitus (DM) inhibe la biosíntesis de serotonina cerebral mediante cambios en la actividad y expresión de triptófano-5-hidroxilasa (TPH). Objetivos: Determinar si los cambios en la expresión de TPH1 y TPH2 cerebral y en el número de neuronas serotoninérgicas causados por la DM retornan a la normalidad en ratas con diabetes tratadas con insulina. Métodos: Ratas con diabetes inducida con estreptozotocina se dividieron en dos grupos uno tratado con insulina y otro sin tratamiento. En el día 14, se obtuvieron tallos cerebrales para cuantificar niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH. La expresión de TPH1 y TPH2 fue mediante Western blot y el número de neuronas serotoninérgicas por inmunohistoquímica. Resultados: En las ratas con diabetes se confirmó disminución de los niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH, así como menor expresión de TPH1 y TPH2 y menor número de neuronas serotoninérgicas. Cuando las ratas diabéticas fueron tratadas con insulina, el L-triptófano regresó a la normalidad, no así la 5-hidroxitriptamina, la expresión de TPH ni el número de neuronas serotoninérgicas. Conclusiones: La DM inhibe crónicamente la síntesis de 5-hidroxitriptamina cerebral mediante modificaciones en la expresión de TPH1 y TPH2 y disminución de las neuronas serotoninérgicas, que persisten a pesar del tratamiento con insulina.

Abstract Introduction: Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. Objectives: To determine whether DM-induced changes in brain TPH1 and TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. Methods: Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expressión of TPH1 and THP2 by Western blot, and the number of serotonergic neurons by immunohistochemistry. Results: In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hydroxytryptamine, TPH expression, or the number of serotonergic neurons. Conclusions: DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions.

INTRODUCTION: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. OBJECTIVE: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). METHODS: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. RESULTS: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). CONCLUSIONS: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

INTRODUCCIÓN: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. OBJETIVO: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). MÉTODOS: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. RESULTADOS: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). CONCLUSIONES: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Structural optimization of 3D-printed patient-specific ceramic scaffolds for in vivo bone regeneration in load-bearing defects.

Tissue engineering has recently gained popularity as an alternative to autografts to stimulate bone tissue regeneration through structures called scaffolds. Most of the in vivo experiments on long-bony defects use internally-stabilized generic scaffolds. Despite the wide variety of computational methods, a standardized protocol is required to optimize ceramic scaffolds for load-bearing bony defects stabilized with flexible fixations. An optimization problem was defined for applications to sheep metatarsus defects. It covers biological parameters (porosity, pore size, and the specific surface area) and mechanical constraints based on in vivo and in vitro results reported in the literature. The optimized parameters (59.30% of porosity, 5768.91 m-1 of specific surface area, and 360.80 µm of pore size) and the compressive strength of the selected structure were validated in vitro by means of tomographic images and compression tests of six 3D-printed samples. Divergences between the design and measured values of the optimized parameters, mainly due to manufacturing defects, are consistent with the previous studies. Using the mixed experimental-mathematical scaffold-design procedure described, they could be implanted in vivo with instrumented external fixators, therefore facilitating biomechanical monitoring of the regeneration process.

Mechanical Influence of Surrounding Soft Tissue on Bone Regeneration Processes: A Bone Lengthening Study.

Bone lengthening is a bone regeneration technique with multiple clinical applications. One of the most common complications of this treatment is the lack of adaptation of the surrounding soft tissue to their extension. A better understanding of the mechanobiology of the tissues involved in distraction osteogenesis would allow better control of the clinical cases. Bone lengthening treatments were performed in vivo in the metatarsus of Merino sheep, measuring the distraction forces by means of an instrumented fixator. The tissue relaxation after distraction was analyzed in this study. A viscoelastic model was also applied to distraction data to assess the mechanical behavior of the tissues during the distraction phase. Tissue relaxation is similar to other bone regeneration processes which do not imply surrounding soft tissue extension, e.g. bone transport. The effects of this tissue on distraction forces are limited to the first minutes of distraction and elongations above 4% of the original length with the protocol applied. Moreover, the surrounding soft tissue initially loses some of its viscoelasticity and subsequently suffers strain hardening from day 5 of distraction until the end of the distraction phase, day 15. Finally, anatomical changes were also evidenced in the elongated limb of our specimens.

Mathematical formulation and parametric analysis of in vitro cell models in microfluidic devices: application to different stages of glioblastoma evolution.

In silico models and computer simulation are invaluable tools to better understand complex biological processes such as cancer evolution. However, the complexity of the biological environment, with many cell mechanisms in response to changing physical and chemical external stimuli, makes the associated mathematical models highly non-linear and multiparametric. One of the main problems of these models is the determination of the parameters' values, which are usually fitted for specific conditions, making the conclusions drawn difficult to generalise. We analyse here an important biological problem: the evolution of hypoxia-driven migratory structures in Glioblastoma Multiforme (GBM), the most aggressive and lethal primary brain tumour. We establish a mathematical model considering the interaction of the tumour cells with oxygen concentration in what is called the go or grow paradigm. We reproduce in this work three different experiments, showing the main GBM structures (pseudopalisade and necrotic core formation), only changing the initial and boundary conditions. We prove that it is possible to obtain versatile mathematical tools which, together with a sound parametric analysis, allow to explain complex biological phenomena. We show the utility of this hybrid "biomimetic in vitro-in silico" platform to help to elucidate the mechanisms involved in cancer processes, to better understand the role of the different phenomena, to test new scientific hypotheses and to design new data-driven experiments.

[Anatomopathological findings during development of diabetic cardiomyopathy in rats]. / Hallazgos anatomopatológicos durante el desarrollo de la miocardiopatía diabética en la rata.

BACKGROUND: The diabetic cardiomyopathy occurs in both type 1 and type 2 diabetes mellitus. Hyperglycemia and associated metabolic changes participate in the pathogenesis of this disease. OBJECTIVE: To characterizes various pathological changes occurring during the development of diabetic cardiomyopathy in rats. METHODS: Diabetic rats were used for streptozotocin administration. At 7, 14, 21 and 30 days after toxic administration, the heart was obtained and placed in a Hartman solution and 4% p-formaldehyde. Five-micrometer thick sections were stained with hematoxylin-eosin, Masson trichrome and immunocytochemistry using anti-ß-tubulin antibody. RESULTS: At 14 days after application of streptozotocin, dilated sinusoids with endothelial lining in the myocardium and collagen deposits in the cardiac interstitium and between the Purkinje fibers were observed. At 21 days there was a slight decrease of the arteriolar lumen due to hyperplasia of the medial layer. It is important to note that cardiac sinusoids as well as collagen deposits became more evident at 30 day of the study, as well as a major derangement of the microtubular system of the cardiomyocytes. CONCLUSIONS: Cardiac sinusoids representing fetal vascular pattern and interstitial fibrosis in the myocardium and the microtubular derangement of cardiomyocytes support the fact that the pathophysiological mechanism of diabetic cardiomyopathy begins in the coronary microcirculation due to changes in cardiac metabolism, contributing to the development of myocardial dysfunction in diabetes.

Antecedentes: la miocardiopatía diabética ocurre en ambos tipos de diabetes mellitus y en su patogenia intervienen la hiperglucemia y los cambios metabólicos asociados. Objetivo: caracterizar los diferentes cambios patológicos que aparecen durante la evolución de la miocardiopatía diabética en la rata. Material y métodos: estudio transversal comparativo en dos grupos de ratas diabéticas por la administración de estreptozotocina. A los 14, 21 y 30 días de la administración del tóxico se obtuvieron los corazones, que se colocaron en p-formaldehído al 4%. Se efectuaron cortes de 5 µm y se tiñeron con hematoxilina-eosina, tricrómica de Masson e inmunocitoquímica con anticuerpos anti ß-tubulina. Resultados: a los 14 días de la aplicación de la estreptozotocina se observaron en el miocardio sinusoides dilatadas y depósito de colágena entre las fibras de Purkinje e intersticio cardiaco. A los 21 días disminuyó la luz arteriolar por hiperplasia de la capa media. A los 30 días del estudio se hicieron más evidentes los sinusoides cardiacos y los depósitos de colágena y un importante desarreglo del sistema microtubular de los cardiomiocitos. Conclusiones: los sinusoides cardiacos, que representan un patrón vascular fetal y la fibrosis intersticial en el miocardio y el desarreglo microtubular de los cardiomiocitos, apoyan el hecho de que el mecanismo fisiopatológico de la miocardiopatía diabética se inicia en la microcirculación coronaria debido a cambios en el metabolismo cardiaco que contribuyen a la disfunción miocárdica durante el estado diabético.

Comparative evaluation of the effectiveness and tolerability of nimesulide versus rofecoxib taken once a day in the treatment of patients with knee osteoarthritis.

UNLABELLED: The treatment of osteoarthritis, a common degenerative joint disease, is focused on the relief of symptoms, mainly pain, and sometimes joint stiffness. Nonsteroidal antiinflammatory drugs are considered the treatment of choice in cases where the pain cannot be reduced with acetaminophen. This trial was conducted to compare the effectiveness of 2 nonsteroidal antiinflammatory drugs taken once daily to relieve the symptoms of osteoarthritis. OBJECTIVE: To compare the safety and efficacy of 2 nonsteroidal antiinflammatory drugs, nimesulide versus rofecoxib, given once a day in patients with knee osteoarthritis. METHODS: 114 patients with the diagnosis of knee osteoarthritis were recruited in a randomized, double-blinded fashion, and they received a daily dose of either 300 mg of nimesulide retard or 25 mg of rofecoxib. The effectiveness was evaluated by means of: visual analog scale 0 to 10 cm measured at 0, 15, 30, and 45 minutes after the first dose and then after 2, 3, 15, and 30 days of continuous drug dosing. The quality of life was assessed by the scale of the Western Ontario and McMaster Universities on days 0, 15, and 30. The patients' opinions and that of the physicians were recorded at the end of the study. RESULTS: The 2 groups of patients were matched for race, sex, menopause status (for females), pathologic antecedents, the duration of osteoarthritis, and the history of nonsteroidal antiinflammatory drug therapy; however, patients' affected knees could differ in the 2 treatments. The 2 drugs were equally effective in terms of overall improvement of pain and the quality of life; they were equally well tolerated. However, significant differences were found between the 2 drugs in favor of nimesulide in the relief of the pain on days 2, 3, and 30, and in the scale of Western Ontario and McMaster Universities Osteoarthritis index on day 15 and 30. DISCUSSION: In this trial, both medications were effective in improving the pain and the quality of life in patients with knee osteoarthritis; however, nimesulide, compared with rofecoxib, was quicker in reducing the pain and was more effective on days 2, 3, and 30. Nimesulide was also superior in improving the quality of life after 30 days of treatment.

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa.

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.