Reverse Phase HPLC Methodology for the Determination of Bay K8644.

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2 mL/min. Mean retention time for Bay K8644 was 3.030 minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40 µg/mL at 274 nm wavelength. Analytical limits were 16.56 ± 1.04 ng/mL for detection and 55.21 ± 3.48 ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4 minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.

Aportes del Hospital de Día para la atención de niños/as y adolescentes con patologías mentales graves, en la formación de residentes de Psiquiatría Pediátrica / Contributions from the Day Hospital for the care of children and adolescents with serious mental pathologies, in the training of Pediatric Psychiatry residents

Cerca del 20 % de niños en todo el mundo experimentan trastornos mentales, independientemente de su procedencia o cultura. La manifestación de un trastorno mental durante la infancia puede ocasionar alteraciones en el desarrollo, impactando negativamente en la calidad de vida, la dinámica familiar, y en el progreso académico y social. En nuestro país los problemas de salud mental en la infancia y adolescencia están entre los principales problemas de salud en este grupo de edades, y las consultas y hospitalizaciones de niños, niñas y adolescentes se encuentran con una oferta de servicios limitada. En 2017, se aprueba la Ley de Salud Mental, que promueve la atención de los problemas en el ámbito comunitario, y la implementación de distintos dispositivos de atención alternativos a la hospitalización a tiempo completo. Uno de los dispositivos intermedios es el hospital de día, para la atención de trastornos mentales en forma ambulatoria diurna, como alternativa a la hospitalización. En el 2021 se abre el primer Hospital de Día para niños, niñas y adolescentes con trastornos psiquiátricos severos en el Centro Hospitalario Pereira Rossell. Con un equipo interdisciplinario con recursos de la Administración de los Servicios de Salud del Estado y la Facultad de Medicina, brinda la oportunidad de pasantías para estudiantes de distintas disciplinas. El Hospital de Día enriquece la formación de residentes de Psiquiatría Pediátrica para enfrentar desafíos clínicos y desarrollar estrategias de tratamiento interdisciplinarias, complementarias al abordaje tradicional, una atención de mayor calidad y una oportunidad de aprendizaje en trabajo en equipo.

About 20% children experience mental disorders worldwide, regardless of background or culture. Mental disorders appearing during childhood can cause developmental changes, with negative impact on quality of life, family dynamics, as well as on academic and social progress. In our country, Mental Health problems in childhood and adolescence are among the main health problems in this age group, increasing consultations and hospitalizations of children and adolescents, stressing a limited supply of services. In 2017, the Mental Health Law was passed, which promotes mental health care at the community level, and the implementation of different care strategies as alternatives to fulltime hospitalization. One of the intermediate strategies is the day hospital, for the care of mental disorders on a daytime outpatient basis, as an alternative to conventional hospitalization. In 2021, the first day hospital for children and adolescents with severe psychiatric disorders was opened in Uruguay at the Pereira Rossell Hospital Center. With an interdisciplinary team with resources from ASSE and the Faculty of Medicine, it provides opportunity for internships for students from different disciplines. The Day Hospital enriches the training of Pediatric Psychiatry residents. They learn how to cope with clinical challenges, have a teamwork learning opportunity, and develop interdisciplinary treatment strategies, complementary to the traditional approach, providing higher quality care in the management of serious childhood mental disorders.

Increased proliferation and neuronal fate in prairie vole brain progenitor cells cultured in vitro: effects by social exposure and sexual dimorphism.

BACKGROUND: The prairie vole (Microtus ochrogaster) is a socially monogamous rodent that establishes an enduring pair bond after cohabitation, with (6 h) or without (24 h) mating. Previously, we reported that social interaction and mating increased cell proliferation and differentiation to neuronal fate in neurogenic niches in male voles. We hypothesized that neurogenesis may be a neural plasticity mechanism involved in mating-induced pair bond formation. Here, we evaluated the differentiation potential of neural progenitor cells (NPCs) isolated from the subventricular zone (SVZ) of both female and male adult voles as a function of sociosexual experience. Animals were assigned to one of the following groups: (1) control (Co), sexually naive female and male voles that had no contact with another vole of the opposite sex; (2) social exposure (SE), males and females exposed to olfactory, auditory, and visual stimuli from a vole of the opposite sex, but without physical contact; and (3) social cohabitation with mating (SCM), male and female voles copulating to induce pair bonding formation. Subsequently, the NPCs were isolated from the SVZ, maintained, and supplemented with growth factors to form neurospheres in vitro. RESULTS: Notably, we detected in SE and SCM voles, a higher proliferation of neurosphere-derived Nestin + cells, as well as an increase in mature neurons (MAP2 +) and a decrease in glial (GFAP +) differentiated cells with some sex differences. These data suggest that when voles are exposed to sociosexual experiences that induce pair bonding, undifferentiated cells of the SVZ acquire a commitment to a neuronal lineage, and the determined potential of the neurosphere is conserved despite adaptations under in vitro conditions. Finally, we repeated the culture to obtain neurospheres under treatments with different hormones and factors (brain-derived neurotrophic factor, estradiol, prolactin, oxytocin, and progesterone); the ability of SVZ-isolated cells to generate neurospheres and differentiate in vitro into neurons or glial lineages in response to hormones or factors is also dependent on sex and sociosexual context. CONCLUSION: Social interactions that promote pair bonding in voles change the properties of cells isolated from the SVZ. Thus, SE or SCM induces a bias in the differentiation potential in both sexes, while SE is sufficient to promote proliferation in SVZ-isolated cells from male brains. In females, proliferation increases when mating is performed. The next question is whether the rise in proliferation and neurogenesis of cells from the SVZ are plastic processes essential for establishing, enhancing, maintaining, or accelerating pair bond formation. Highlights 1. Sociosexual experiences that promote pair bonding (social exposure and social cohabitation with mating) induce changes in the properties of neural stem/progenitor cells isolated from the SVZ in adult prairie voles. 2. Social interactions lead to increased proliferation and induce a bias in the differentiation potential of SVZ-isolated cells in both male and female voles. 3. The differentiation potential of SVZ-isolated cells is conserved under in vitro conditions, suggesting a commitment to a neuronal lineage under a sociosexual context. 4. Hormonal and growth factors treatments (brain-derived neurotrophic factor, estradiol, prolactin, oxytocin, and progesterone) affect the generation and differentiation of neurospheres, with dependencies on sex and sociosexual context. 5. Proliferation and neurogenesis in the SVZ may play a crucial role in establishing, enhancing, maintaining, or accelerating pair bond formation.

In this study, researchers evaluated whether social interactions and copulation induce changes in the proliferation and differentiation of neural progenitor cells in adult male and female voles using an in vitro neurosphere formation assay. The following groups were assigned: control animals without any exposure to another vole outside their litter, another group with social exposure consisting of sensory exposure to a vole of the opposite sex and a third group with social cohabitation and copulation. Forty eight hours after social interactions, cells were isolated from the neurogenic niche subventricular zone (SVZ) and cultured to assess their self-renewal and proliferation abilities to form neurospheres. The results showed in the social interaction groups, a greater number and growth of neurospheres in both males and females. Differentiation capacity was assessed by immunodetection of MAP2 and GFAP to identify neurons or glia, respectively, arise from neurospheres, with an increase in neuronal fate in groups with social interaction. In the second part of the study, the researchers analyzed the effect of different hormone and growth factor treatments and found that the response in both proliferation and differentiation potential may vary depending on the sociosexual context or sex. This study suggests that social interactions leading to pair bond formation alter the properties of SVZ cells, whereby proliferation and neurogenesis may have an impact on the establishment and maintenance of pair bonding.

Pair-bonding and social experience modulate new neurons survival in adult male and female prairie voles (Microtus ochrogaster).

Prairie voles are a socially monogamous species that, after cohabitation with mating, form enduring pair bonds. The plastic mechanisms involved in this social behavior are not well-understood. Neurogenesis in adult rodents is a plastic neural process induced in specific brain areas like the olfactory bulbs (OB) and dentate gyrus (DG) of the hippocampus. However, it is unknown how cell survival is modulated by social or sexual experience in prairie voles. This study aimed to evaluate if cohabitation with mating and/or social exposure to a vole of the opposite sex increased the survival of the new cells in the main and accessory OB and DG. To identify the new cells and evaluate their survival, voles were injected with the DNA synthesis marker 5-bromo-2'-deoxyuridine (BrdU) and were randomly distributed into one of the following groups: (A) Control (C), voles that did not receive any sexual stimulation and were placed alone during the behavioral test. (B) Social exposure (SE), voles were individually placed in a cage equally divided into two compartments by an acrylic screen with small holes. One male and one female were placed in opposite compartments. (C) Social cohabitation with mating (SCM), animals mated freely. Our findings demonstrated that SCM females had increases in the number of new cells (BrdU-positive cells) in the main olfactory bulb and new mature neurons (BrdU/NeuN-positive cells) in the glomerular layer (GlL). In contrast, these new cells decrease in males in the SE and SCM conditions. In the granular cell layer (GrL), SCM females had more new cells and neurons than the SE group. In the accessory olfactory bulb, in the anterior GlL, SCM decreased the number of new cells and neurons in females. On the other hand, in the DG, SCM and SE increase the number of new cells in the suprapyramidal blade in female voles. Males from SCM express more new cells and neurons in the infrapyramidal blade compared with SE group. Comparison between male and females showed that new cells/neurons survival was sex dependent. These results suggest that social interaction and sexual behavior modulate cell survival and influence the neuronal fate in a sex-dependent manner, in the OB and DG. This study will contribute to understand neural mechanisms of complex social and pair bond behaviors in the prairie voles; supporting adult neurogenesis as a plastic mechanism potentially involved in social monogamous strategy.

Docetaxel in chitosan-based nanocapsules conjugated with an anti-Tn antigen mouse/human chimeric antibody as a promising targeting strategy of lung tumors.

The aim of this work was to evaluate the physicochemical and biological properties of docetaxel (DCX) loaded chitosan nanocapsules (CS Nc) functionalized with the monoclonal antibody Chi-Tn (CS-PEG-ChiTn mAb Nc) as a potential improvement treatment for cancer therapy. The Tn antigen is highly specific for carcinomas, and this is the first time that such structure is targeted for drug delivery. The nanocapsules (Ncs), formed as a polymeric shell around an oily core, allowed a 99.9% encapsulation efficiency of DCX with a monodispersity particle size in the range of 200 nm and a high positive surface charge that provide substantial stability to the nanosystems. Release profile of DCX from Ncs showed a sustained and pH dependent behavior with a faster release at acidic pH, which could be favorable in the intracellular drug delivery. We have designed PEGylated CS Nc modified with a monoclonal antibody which recognize Tn antigen, one of the most specific tumor associated antigen. A biotin-avidin approach achieved the successful attachment of the antibody to the nanocapsules. Uptake studies and viability assay conducted in A549 human lung cancer cell line in vitro demonstrate that ChiTn mAb enhance nanoparticles internalization and cell viability reduction. Consequently, these ChiTn functionalized nanocapsules are promising carriers for the active targeting of DCX to Tn expressing carcinomas.

Physico-chemical and antilisterial properties of nisin-incorporated chitosan/carboxymethyl chitosan films.

The objective of this work was to investigate the effect of the addition of carboxymethyl chitosan on the structural properties and antilisterial activity of nisin-incorporated chitosan films. Chitosan and carboxymethyl chitosan solutions were prepared with different mass ratios and bacteriocin nisin was added (0, 1000 and 6000 IU/ml). Filmogenic solutions were cast, dried and their physico-chemical and antimicrobial properties were investigated. For the same chitosan/carboxymethyl chitosan mass ratio, the addition of NIS at 6000 IU/ml led to changes in the macro- and microstructure, as well as in physico-chemical properties of films. On the other hand, carboxymethyl chitosan had a plasticizing effect and enhanced the distribution of the bacteriocin within the biopolymer matrix. Moreover, nisin-incorporated blend films of chitosan and carboxymethyl chitosan were more effective against Listeria monocytogenes than their pure chitosan counterparts. This study showed that different formulations of nisin-incorporated composite films of chitosan and carboxymethyl chitosan may provide options for developing bioactive packaging to improve food safety.

Clinical impact and cost analysis of the use of either the Xpert MTB Rif test or sputum smear microscopy in the diagnosis of pulmonary tuberculosis in Rio de Janeiro, Brazil.

INTRODUCTION: The molecular test Xpert MTB/RIF (Xpert) has been recommended for use in the diagnosis of pulmonary tuberculosis (PTB); however, data on the cost of incorporating it under routine conditions in high-burden countries are scarce. The clinical impact and costs incurred in adopting the Xpert test in routine PTB diagnosis was evaluated in a prospective study conducted from November 2012 to November of 2013, in the City of Rio de Janeiro, Brazil. METHODS: The diagnostic and therapeutic cascade for TB treatment was evaluated using Xpert in the first stage (S1), and sputum smear microscopy (SSM) in the second stage (S2). The mean costs associated with each diagnostic test were calculated including equipment, human resources, supplies, and infrastructure. RESULTS: We included 232 subjects with probable TB (S1 = 87; S2 = 145). The sensitivities of Xpert and SSM were 91.7% (22/24) and 79.1% (34/43), respectively. The median time between triage and TB treatment initiation in S1 (n = 24) was 14.5 days (IQR 8-28.0) and in S2 (n = 43) it was 8 days [interquartile range (IQR) 6-12.0]. The estimated mean costs per examination in S1 and S2 were US$24.61 and US$6.98, respectively. CONCLUSIONS: Compared with SSM, Xpert test showed a greater sensitivity, but it also had a time delay with respect to treatment initiation and a higher mean cost per examination.

Clinical impact and cost analysis of the use of either the Xpert MTB Rif test or sputum smear microscopy in the diagnosis of pulmonary tuberculosis in Rio de Janeiro, Brazil

Abstract INTRODUCTION: The molecular test Xpert MTB/RIF (Xpert) has been recommended for use in the diagnosis of pulmonary tuberculosis (PTB); however, data on the cost of incorporating it under routine conditions in high-burden countries are scarce. The clinical impact and costs incurred in adopting the Xpert test in routine PTB diagnosis was evaluated in a prospective study conducted from November 2012 to November of 2013, in the City of Rio de Janeiro, Brazil. METHODS: The diagnostic and therapeutic cascade for TB treatment was evaluated using Xpert in the first stage (S1), and sputum smear microscopy (SSM) in the second stage (S2). The mean costs associated with each diagnostic test were calculated including equipment, human resources, supplies, and infrastructure. RESULTS: We included 232 subjects with probable TB (S1 = 87; S2 = 145). The sensitivities of Xpert and SSM were 91.7% (22/24) and 79.1% (34/43), respectively. The median time between triage and TB treatment initiation in S1 (n = 24) was 14.5 days (IQR 8-28.0) and in S2 (n = 43) it was 8 days [interquartile range (IQR) 6-12.0]. The estimated mean costs per examination in S1 and S2 were US$24.61 and US$6.98, respectively. CONCLUSIONS: Compared with SSM, Xpert test showed a greater sensitivity, but it also had a time delay with respect to treatment initiation and a higher mean cost per examination.

Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and ß-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

Hypoxic preconditioning differentially affects GABAergic and glutamatergic neuronal cells in the injured cerebellum of the neonatal rat.

In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult--showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization--were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are protected by a preconditioning hypoxia while the latter are not.

Patients' costs and cost-effectiveness of tuberculosis treatment in DOTS and non-DOTS facilities in Rio de Janeiro, Brazil.

BACKGROUND: Costs of tuberculosis diagnosis and treatment may represent a significant burden for the poor and for the health system in resource-poor countries. OBJECTIVES: The aim of this study was to analyze patients' costs of tuberculosis care and to estimate the incremental cost-effectiveness ratio (ICER) of the directly observed treatment (DOT) strategy per completed treatment in Rio de Janeiro, Brazil. METHODS: We interviewed 218 adult patients with bacteriologically confirmed pulmonary tuberculosis. Information on direct (out-of-pocket expenses) and indirect (hours lost) costs, loss in income and costs with extra help were gathered through a questionnaire. Healthcare system additional costs due to supervision of pill-intake were calculated considering staff salaries. Effectiveness was measured by treatment completion rate. The ICER of DOT compared to self-administered therapy (SAT) was calculated. PRINCIPAL FINDINGS: DOT increased costs during the treatment phase, while SAT increased costs in the pre-diagnostic phase, for both the patient and the health system. Treatment completion rates were 71% in SAT facilities and 79% in DOT facilities. Costs per completed treatment were US$ 194 for patients and U$ 189 for the health system in SAT facilities, compared to US$ 336 and US$ 726 in DOT facilities. The ICER was US$ 6,616 per completed DOT treatment compared to SAT. CONCLUSIONS: Costs incurred by TB patients are high in Rio de Janeiro, especially for those under DOT. The DOT strategy doubles patients' costs and increases by fourfold the health system costs per completed treatment. The additional costs for DOT may be one of the contributing factors to the completion rates below the targeted 85% recommended by WHO.

A standardized surgical technique for rat superior cervical ganglionectomy.

Superior cervical ganglionectomy (SCGx) is a valuable microsurgical model to study the role of the sympathetic nervous system in a vast array of physiological and pathological processes, including homeostatic regulation, circadian biology and the dynamics of neuronal dysfunction and recovery after injury. Despite having several experimental applications in the rat, a thorough description of a standardized procedure has never been published. Here, we provide a brief review of the principal features and experimental uses of the SCGx, the surgical anatomy of the neck and sympathetic cervical chain, and a step-by-step description of how to consistently remove the superior cervical ganglia through the omohyoid muscle or the carotid triangle. Furthermore, we suggest procedures and precautions to be taken during and after surgery to optimize results and describe tools to validate surgical success. We expect that the following standardized and optimized protocol will allow researchers to organize knowledge into a cohesive framework in those areas where the SCGx is applied.

Abnormal production of transforming growth factor beta and interferon gamma by peripheral blood cells of patients with multidrug-resistant pulmonary tuberculosis in Brazil.

OBJECTIVES: The aim of the present study was to determine the immune response profile that differentiates patients with newly diagnosed (non-treated) pulmonary tuberculosis from multidrug-resistant (MDR) ones, as well as from healthy, tuberculin positive individuals. METHODS: Lymphocytes proliferative response to non-specific mitogen (PHA) and PPD were evaluated by 3H thymidine incorporation and cytokines were quantified using an ELISA assay. RESULTS: Patients with active disease showed a diminished proliferative response to PHA and PPD, while multidrug-resistant patients showed a diminished proliferative response to PHA, but a normal response to PPD. The cytokine production of newly diagnosed patients was characterized by a diminished production of IFNgamma and normal production of transforming growth factor (TGFbeta), while MDR patients revealed a normal production of IFNgamma accompanied by an increase in TGFbeta. CONCLUSIONS: The production of significant amounts of TGFbeta in MDR patients leads to a poor immune response and may contribute to the resistance of tuberculosis patients to drugs.