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Hemophilia patients enrolled in clinical trials often exhibit various physical and psychological symptoms. However, little is known about anxiety and depression among them. This study assessed the effects of depression and anxiety on hemophilia patients enrolled in clinical trials and identified risk factors for these disorders. A multi-center, cohort study was conducted from January to December 2022. Sixty-nine hemophilia patients who enrolled in clinical trials participated at baseline (T1, prior to treatment initiation) and completed the informed consent. Risk factors were measured at baseline to predict depression and anxiety at 3 months (T2). Sixty-four hemophilia patients were included in the final analysis. More hemophilia patients had moderate-severe depression (28 [43.75%]) and anxiety (16 [25.00%]) at T2 than at T1 (12 [18.75]), (5 [7.81%]). Depression was aggravated in 23 (35.94%) patients and anxiety was aggravated in 12 (18.75%) patients. Frequently acquired medical information (OR 11.378, CI 1.319-98.114, P = 0.027), baseline GAD-7 (OR 1.341, CI 1.015-1.772, P = 0. 039) and PHQ-9 (OR 1.465, CI 1.039-2.065, P = 0.029) are important factors predicting depression and anxiety in hemophilia patients. Hemophilia patients enrolled in a clinical trial have significant anxiety and depression. The frequency of acquiring medical information and the baseline PHQ-9 and GAD-7 scores were risk factors for anxiety and depression. Thus, hemophilia patients should receive education regarding clinical trials and undergo evaluations of their anxiety and depression; these changes will enable prompt detection of their psychological burden and identify potential psychological intervention strategies.
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Depresión , Hemofilia A , Humanos , Depresión/psicología , Estudios de Cohortes , Ansiedad/psicologíaRESUMEN
PURPOSE: The histopathological study of brain tissue is a common method in neuroscience. However, efficient procedures to preserve the intact hypothalamic-pituitary brain specimens are not available in mice for histopathological study. METHOD: We describe a detailed procedure for obtaining mouse brain with pituitary-hypothalamus continuity. Unlike the traditional methods, we collect the brain via a ventral approach. We cut the intraoccipital synchondrosis, transection the endocranium of pituitary, broke the spheno-occipital synchondrosis, expose the posterior edge of pituitary, separate the trigeminal nerve, then the intact pituitary gland was preserved. RESULT: We report an more effective and practical method to obtain continuous hypothalamus -pituitary preparations based on the preserve of leptomeninges. COMPARED WITH THE EXISTING METHODS: Our procedure effectively protects the integrity of the fragile infundibulum preventing the pituitary from separating from the hypothalamus. This procedure is more convenient and efficient. CONCLUSION: We present a convenient and practical procedure to obtain intact hypothalamic-pituitary brain specimens for subsequent histopathological evaluation in mice.
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Enfermedades de la Hipófisis , Neurohipófisis , Ratones , Animales , Hipófisis/patología , Neurohipófisis/patología , Hipotálamo/patología , Sistema Hipotálamo-Hipofisario , Enfermedades de la Hipófisis/cirugía , Enfermedades de la Hipófisis/patologíaRESUMEN
Body fluid homeostasis is critical to survival. The integrity of the hypothalamo-neurohypophysial system (HNS) is an important basis of the precise regulation of body fluid metabolism and arginine vasopressin (AVP) hormone release. Clinically, some patients with central diabetes insipidus (CDI) due to HNS lesions can experience recovery compensation of body fluid metabolism. However, whether the hypothalamus has the potential for structural plasticity and self-repair under pathological conditions remains unclear. Here, we report the repair and reconstruction of a new neurohypophysis-like structure in the hypothalamic median eminence (ME) after pituitary stalk electrical lesion (PEL). We show that activated and proliferating adult neural progenitor cells differentiate into new mature neurons, which then integrate with remodeled AVP fibers to reconstruct the local AVP hormone release neural circuit in the ME after PEL. We found that the transcription factor of NK2 homeobox 1 (NKX2.1) and the sonic hedgehog signaling pathway, mediated by NKX2.1, are the key regulators of adult hypothalamic neurogenesis. Taken together, our study provides evidence that adult ME neurogenesis is involved in the structural reconstruction of the AVP release circuit and eventually restores body fluid metabolic homeostasis during hypothalamic self-repair.
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Líquidos Corporales , Eminencia Media , Arginina Vasopresina/metabolismo , Líquidos Corporales/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Hipotálamo/metabolismo , Eminencia Media/metabolismo , Neurogénesis , Hipófisis/metabolismoRESUMEN
BACKGROUND: Hypothalamic injury causes several complicated neuroendocrine-associated disorders, such as water-electrolyte imbalance, obesity, and hypopituitarism. Among these, central diabetes insipidus (CDI), characterized by polyuria, polydipsia, low urine specific gravity, and deficiency of arginine vasopressin contents, is a typical complication after hypothalamic injury. METHODS: CDI was induced by hypothalamic pituitary stalk injury in male animals. Behavioral parameters and blood sample were collected to evaluate the characteristics of body fluid metabolism imbalance. The brains were harvested for high-throughput RNA sequencing and immunostaining to identify pathophysiological changes in corresponding hypothalamic nuclei. RESULTS: Based on transcriptomic analysis, we demonstrated the upregulation of the activating transcription factor 3 (Atf3)/c-Jun axis and identified Lgals3, a microglial activation-related gene, as the most significant target gene in response to the body fluid imbalance in CDI. Furthermore, we found that the microglia possessed elevated phagocytic ability, which could promote the elimination of arginine vasopressin neurons after hypothalamic injury. CONCLUSION: Our findings suggested that the Atf3/c-Jun/Lgals3 axis was associated with the microglial activation, and might participate in the loss of functional arginine vasopressin neurons in CDI after hypothalamic injury.
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Diabetes Insípida Neurogénica , Diabetes Mellitus , Enfermedades Hipotalámicas , Factor de Transcripción Activador 3/metabolismo , Animales , Arginina Vasopresina/metabolismo , Diabetes Insípida Neurogénica/complicaciones , Galectina 3/metabolismo , Enfermedades Hipotalámicas/complicaciones , Masculino , TranscriptomaRESUMEN
Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in the central nervous system (CNS). As the ideal targets for GBM treatment, Src family kinases (SFKs) have attracted much attention. Herein, a new series of imidazo[4,5-c]pyridin-2-one derivatives were designed and synthesised as SFK inhibitors. Compounds 1d, 1e, 1q, 1s exhibited potential Src and Fyn kinase inhibition in the submicromolar range, of which were next tested for their antiproliferative potency on four GBM cell lines. Compound 1s showed effective activity against U87, U251, T98G, and U87-EGFRvIII GBM cell lines, comparable to that of lead compound PP2. Molecular dynamics (MDs) simulation revealed the possible binding patterns of the most active compound 1s in ATP binding site of SFKs. ADME prediction suggested that 1s accord with the criteria of CNS drugs. These results led us to identify a novel SFK inhibitor as candidate for GBM treatment.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinolonas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Imidazoles/síntesis química , Imidazoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Quinolonas/síntesis química , Quinolonas/química , Relación Estructura-Actividad , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Spindle and kinetochore associated protein 1 (SKA1) is a protein involved in chromosome congression and mitosis. It has been found to be upregulated and oncogenic in several human cancers. Herein, we investigated the precise role of SKA1 in the progression and malignant phenotype of human glioma. METHODS: Bioinformatic analysis was carried out based on the RNA-seq data and corresponding clinical data from GEO, TCGA and CGGA databases. Western blot was performed to analyze the expression of SKA1 in clinical samples and signaling pathway proteins in glioma cells, respectively. CCK8 assay, colony forming assay and EdU assay were performed to assess the cell viability. Cell migration and invasion assays were also performed. Moreover, xenograft model was established and the expression of SKA1 was assessed in the xenograft by immunohistochemistry. RESULTS: SKA1 expression is positively correlated with glioma grade and could be a promising biomarker for GBM. Moreover, overexpression of SKA1 may lead to poor prognosis in glioma. Downregulation of SKA1 attenuated cell viability, migration, and invasion in U251, U87, LN229 and T98 cells. Furthermore, GSEA analysis demonstrated that SKA1 was involved in the cell cycle, EMT pathway as well as Wnt/ß-catenin signaling pathway, which were then confirmed with Western blot analysis. CONCLUSION: SKA1 promotes malignant phenotype and progression of glioma via multiple pathways, including cell cycle, EMT, Wnt/ß-catenin signaling pathway. Therefore, SKA1 could be a promising therapeutic target for the treatment of human gliomas.
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As restricted CA-4 analogues, a novel series of [1,2,4]triazolo[1,5-a]pyrimidines possessing 3,4,5-trimethoxylphenyl groups has been achieved successfully via an efficient one-pot three-component reaction of 3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine, 1,3-dicarbonyl compounds and aldehydes. Initial biological evaluation demonstrated some of target compounds displayed potent antitumor activity in vitro against three cancer cell lines. Among them, the most highly active analogue 26 inhibited the growth of HeLa, and A549 cell lines with IC50 values at 0.75, and 1.02⯵M, respectively, indicating excellent selectivity over non-tumoural cell line HEK-293 (IC50â¯=â¯29.94⯵M) which suggested that the target compounds might possess a high safety index. Moreover, cell cycle analysis illustrated that the analogue 26 significantly induced HeLa cells arrest in G2/M phase, meanwhile the compound could dramatically affect cell morphology and microtubule networks. In addition, compound 28 exhibited potent anti-tubulin activity with IC50 values of 9.90⯵M, and molecular docking studies revealed the analogue occupied the colchicine-binding site of tubulin. These observations suggest that [1,2,4]triazolo[1,5-a]pyrimidines represent a new class of tubulin polymerization inhibitors and well worth further investigation aiming to generate potential anticancer agents.
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Antineoplásicos/farmacología , Diseño de Fármacos , Pirimidinas/farmacología , Triazoles/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/química , Moduladores de Tubulina/síntesis química , Moduladores de Tubulina/química , Células Tumorales CultivadasRESUMEN
Established cancer cell lines are routinely used to study cancer. Several factors such as serial passage may affect the reproducibility of experiments with cancer cell lines, but few researches focused on these changes. In the present study, different morphology and decreased tumorigenicity were observed in late passage U87MG cells. In vitro experiments further revealed that late passage U87MG cells possessed lower invasion properties than early passage, whereas no significant differences of proliferation and migration were found between early and late passage U87MG cells. In particular, we confirmed that late passage U87MG cells exhibited more epithelial phenotype with decreased PI3K/Akt pathway and TGF-ß pathway expressions at protein level. In summary, our results focused on the changes of U87MG cells during serial in vitro passage, suggested that passage-induced changes may lead to notable changes of biological characteristics and several molecular transitions in cancer cell lines, indicating the necessity to shorten experiment-span and accomplish experiments with the same or similar passage cancer cell strains.
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Neoplasias Encefálicas/genética , Glioblastoma/genética , Línea Celular Tumoral , Proliferación Celular/genética , Humanos , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
In clinical settings, tumor compression, trauma, surgical injury, and other types of injury can cause hypothalamic damage, resulting in various types of hypothalamic dysfunction. Impaired release of oxytocin can lead to cognitive impairment and affect prognosis and long-term quality of life after hypothalamic injury. Hypothalamic injury-induced cognitive dysfunction was detected in male animals. Behavioral parameters were measured to assess the characteristics of cognitive dysfunction induced by hypothalamic-pituitary stalk lesions. Brains were collected for high-throughput RNA sequencing and immunostaining to identify pathophysiological changes in hippocampal regions highly associated with cognitive function after injury to corresponding hypothalamic areas. Through transcriptomic analysis, we confirmed the loss of oxytocin neurons after hypothalamic injury and the reversal of hypothalamic-induced cognitive dysfunction after oxytocin supplementation. Furthermore, overactivation of the ERK signaling pathway and ß-amyloid deposition in the hippocampal region after hypothalamic injury were observed, and cognitive function was restored after inhibition of ERK signaling pathway overactivation. Our findings suggest that cognitive dysfunction after hypothalamic injury may be caused by ERK hyperphosphorylation in the hippocampal region resulting from a decrease in the number of oxytocin neurons, which in turn causes ß-amyloid deposition.
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Péptidos beta-Amiloides , Disfunción Cognitiva , Hipocampo , Hipotálamo , Sistema de Señalización de MAP Quinasas , Oxitocina , Oxitocina/metabolismo , Oxitocina/farmacología , Animales , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Masculino , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Modelos Animales de Enfermedad , Ratones , FosforilaciónRESUMEN
JOURNAL/nrgr/04.03/01300535-202410000-00026/figure1/v/2024-02-06T055622Z/r/image-tiff Previous studies have shown that growth hormone can regulate hypothalamic energy metabolism, stress, and hormone release. Therefore, growth hormone has great potential for treating hypothalamic injury. In this study, we established a specific hypothalamic axon injury model by inducing hypothalamic pituitary stalk electric lesions in male mice. We then treated mice by intraperitoneal administration of growth hormone. Our results showed that growth hormone increased the expression of insulin-like growth factor 1 and its receptors, and promoted the survival of hypothalamic neurons, axonal regeneration, and vascular reconstruction from the median eminence through the posterior pituitary. Altogether, this alleviated hypothalamic injury-caused central diabetes insipidus and anxiety. These results suggest that growth hormone can promote axonal reconstruction after hypothalamic injury by regulating the growth hormone-insulin-like growth factor 1 axis.
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The hypothalamus is the key region that regulates the neuroendocrine system as well as instinct behaviors, and hypothalamic dysfunction causes refractory clinical problems. Recent studies have indicated that neural stem/progenitor cell (NSPC) in the hypothalamus play a crucial role in hypothalamic function. However, specific hypothalamic NSPC culture methods have not been established, especially not detailed or efficient surgical procedures. The present study presented a convenient, detailed and efficient method for the isolation and cultivation of hypothalamic NSPCs from embryonic day 12.5 mice. The procedure includes embryo acquisition, brain microdissection to quickly obtain hypothalamic tissue and hypothalamic NSPC culture. Hypothalamic NSPCs can be quickly harvested and grow well in both neurosphere and adherent cultures through this method. Additionally, we confirmed the cell origin and evaluated the proliferation and differentiation properties of cultured cells. In conclusion, we present a convenient and practical method for the isolation and cultivation of hypothalamic NSPCs that can be used in extensive hypothalamic studies.
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The homeobox protein cut-like 1 (CUX1) comprises three isoforms and has been shown to be involved in the development of various types of malignancies. However, the expression and role of the CUX1 isoforms in glioma remain unclear. Herein, we first identified that P75CUX1 isoform exhibited consistent expression among three isoforms in glioma with specifically designed antibodies to identify all CUX1 isoforms. Moreover, a significantly higher expression of P75CUX1 was found in glioma compared with non-tumor brain (NB) tissues, analyzed with western blot and immunohistochemistry, and the expression level of P75CUX1 was positively associated with tumor grade. In addition, Kaplan-Meier survival analysis indicated that P75CUX1 could serve as an independent prognostic indicator to identify glioma patients with poor overall survival. Furthermore, CUX1 knockdown suppressed migration and invasion of glioma cells both in vitro and in vivo. Mechanistically, this study found that P75CUX1 regulated epithelial-mesenchymal transition (EMT) process mediated via ß-catenin, and CUX1/ß-catenin/EMT is a novel signaling cascade mediating the infiltration of glioma. Besides, CUX1 was verified to promote the progression of glioma via multiple other signaling pathways, such as Hippo and PI3K/AKT. In conclusion, we suggested that P75CUX1 could serve as a potential prognostic indicator as well as a novel treatment target in malignant glioma.
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Neoplasias Encefálicas/metabolismo , Movimiento Celular , Glioma/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Adolescente , Adulto , Anciano , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Niño , Preescolar , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Isoformas de Proteínas , Proteínas Represoras/genética , Transducción de Señal , Factores de Transcripción/genética , Regulación hacia Arriba , Adulto Joven , beta Catenina/genéticaRESUMEN
A new class of 2-amino-4-(1,2,4-triazol)pyridine derivatives were designed and synthesized as potent epidermal growth factor receptor inhibitors. In particular, compound 10c exhibited significant inhibitory against EGFRL858R/T790M, and also displayed potent anti-proliferative activity against non-small cell lung cancer cell line H1975. Besides, compound 10j showed potent inhibitory activity against glioblastoma cell line U87-EGFRvâ ¢, which was at least 3-fold more potent than Osimertinib and 25-fold superior to Lazertinib. Moreover, molecular modeling and molecular dynamics simulations disclosed the binding model of the most active compound to the domain of EGFR. This contribution provides 2-amino-4-(1,2,4-triazol)pyridines as a new scaffold for EGFRT790M and/or EGFRvâ ¢ inhibitor.
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Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/químicaRESUMEN
Eukaryotic translation elongation factor 1δ (EEF1D), a subunit of the elongation factor 1 complex of proteins, mediates the elongation process of protein synthesis. Besides this canonical role, EEF1D was found overexpressed in many tumors, like hepatocarcinomas and medulloblastomas. In the present study, we demonstrated for the first time that EEF1D may interact with other putative proteins to regulate cell proliferation, migration, and invasion through PI3K/Akt and EMT pathways in glioma. Furthermore, knockdown of EEF1D could reduce cell proliferation and impaired epithelial-mesenchymal transition (EMT) phenotypes, including cell invasion. Taken together, these results indicate that EEF1D and its partner proteins might play a critical role in glioma and serve as a potential therapeutic target of glioma.
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Neoplasias Encefálicas/patología , Movimiento Celular , Transición Epitelial-Mesenquimal , Glioma/patología , Factor 1 de Elongación Peptídica/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glioma/genética , Humanos , Clasificación del Tumor , Invasividad Neoplásica , Factor 1 de Elongación Peptídica/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Opioid addiction is a brain disease that severely harms society and personal health. Although the tremendous numbers of patients worldwide and emerged negative events, effective treatments for opioid addiction are still lacking. Neuropeptide Y (NPY) is one of the main orexigenic peptides that play vital roles in food intake and energy metabolism. However, increasing evidence indicates that NPY may have great potential in mediating reward effects and drug dependence. In the present study, we assessed the expression changes of NPY in the nucleus accumbens at different timepoints following morphine conditioned place preference (CPP) and investigated the functional importance of potential NPY changes. Our results showed that NPY expression significantly decreased in the nucleus accumbens shell (AcbSh) immediately after chronic morphine exposure. Subsequently, it increased rapidly at first and then gradually returned to normal levels. Further data indicated that these NPY changes were involved in morphine reward memory, demonstrated by a reduction in the extinction period after blocking of the Y5 receptor by L-152,804 in the AcbSh and a prolonged duration of the extinction period following the application of NPY. More importantly, the additional results revealed that L-152,804 also remarkably suppressed the reinstatement of morphine CPP. Together, our results indicate that a complicated plasticity of the NPY pathway in AcbSh occurs following morphine CPP, and this plasticity plays an important role in modulating morphine reward memory. These findings may enhance our understanding of the role of the NPY system in opioid addiction and indicate a promising target for opioid addiction treatment.
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Dependencia de Morfina/psicología , Morfina/farmacología , Narcóticos/farmacología , Neuropéptido Y/efectos de los fármacos , Animales , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Psicológico/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Masculino , Plasticidad Neuronal/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/antagonistas & inhibidores , RecompensaRESUMEN
Alternative splicing (AS) is a ubiquitous mechanism in which pre-mRNA can be spliced into divergent variants and involved in carcinogenesis and progression in several cancers. In the present study, we systematically profiled prognostic AS signatures involving both low grade glioma (LGG) and glioblastoma (GBM) and investigated the association of AS signatures with tumor grade and IDH1 status in glioma. Percent spliced in (PSI) values and corresponding clinical data were obtained from TCGA SpliceSeq and TCGA data portal, respectively. Prognostic AS signatures were identified using univariate and stepwise multivariate Cox regression. Heatmap analysis was performed based on prognostic AS signatures. A prognostic signature was established with 69 and 88 AS events, including specific splicing events of MUTYH, STEAP3, and CTNNB1, in LGG and GBM cohorts, respectively. The area under the curve (AUC) of the prediction model was 0.968 at 2000 days of overall survival (OS) in the LGG cohort and 0.966 at 450 days of OS in the GBM cohort. In addition, these prognostic AS signatures could complement current molecular classification, such as IDH1 mutation, 1p/19q codeletion, and ATRX loss, of glioma and further identify potential subgroups of glioma with the same molecular features. In conclusion, our study systematically profiled prognostic AS events involving both low grade glioma and glioblastoma for the first time, which also shed light on the crosstalk between AS signatures and molecular features of glioma.
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Empalme Alternativo , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Transcriptoma , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Glioma/genética , Glioma/patología , Humanos , Isocitrato Deshidrogenasa/genética , Mutación , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Proteína Nuclear Ligada al Cromosoma X/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: The histopathological study of brain tissue is a conventional method in neuroscience. However, procedures specifically developed to recover intact hypothalamic-pituitary brain specimens, are not available. NEW METHOD: We describe a detailed protocol for obtaining intact rat brain with pituitary-hypothalamus continuity through an intact infundibulum. The brain is collected via a ventral approach through removing the skull base. Membranous structures surrounding the hypothalamus-pituitary system can be preserved, including vasculature. RESULTS: We report a retaining sphenoid and dura technique to obtain intact hypothalamic-pituitary brain preparations, and we confirm the practicability of this method. By combination of this technique with histological analysis or 3D brain tissue clearing and imaging methods, the functional morphology structure of the hypothalamus-pituitary can be further explored. COMPARISON WITH EXISTING METHOD: The current procedure is limited in showing the connection between the hypothalamus and the pituitary. Our procedure effectively protects the integrity of the fragile infundibulum and thus prevents the pituitary from separating from the hypothalamus. CONCLUSIONS: We present a convenient and practical approach to obtain intact hypothalamus-pituitary brain specimens for subsequent histopathological evaluation.
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Hipotálamo , Hipófisis , Animales , Encéfalo/diagnóstico por imagen , RatasRESUMEN
Heterogeneity is regarded as the major factor leading to the poor outcomes of glioblastoma (GBM) patients. However, conventional two-dimensional (2D) analysis methods, such as immunohistochemistry and immunofluorescence, have limited capacity to reveal GBM spatial heterogeneity. Thus, we sought to develop an effective analysis strategy to increase the understanding of GBM spatial heterogeneity. Here, 2D and three-dimensional (3D) analysis methods were compared for the examination of cell morphology, cell distribution and large intact structures, and both types of methods were employed to dissect GBM spatial heterogeneity. The results showed that 2D assays showed only cross-sections of specimens but provided a full view. To visualize intact GBM specimens in 3D without sectioning, the optical tissue clearing methods CUBIC and iDISCO+ were used to clear opaque specimens so that they would become more transparent, after which the specimens were imaged with a two-photon microscope. The 3D analysis methods showed specimens at a large spatial scale at cell-level resolution and had overwhelming advantages in comparison to the 2D methods. Furthermore, in 3D, heterogeneity in terms of cell stemness, the microvasculature, and immune cell infiltration within GBM was comprehensively observed and analysed. Overall, we propose that 2D and 3D analysis methods should be combined to provide much greater detail to increase the understanding of GBM spatial heterogeneity.
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Glioblastoma , Glioblastoma/diagnóstico por imagen , Humanos , Microscopía , Microvasos , FotonesRESUMEN
OBJECTIVE: To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL). METHODS: Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (n=25) and sham operation group (n=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of ß-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting. RESULTS: After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 (P < 0.05) and 7 (P < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 (P < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL. CONCLUSIONS: JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.
Asunto(s)
Arginina Vasopresina/farmacología , Citoesqueleto/metabolismo , Sistema de Señalización de MAP Quinasas , Neuronas/citología , Hipófisis/lesiones , Regeneración , Animales , Apoptosis , Proteína Doblecortina , Hipófisis/citología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Tubulina (Proteína)/metabolismoRESUMEN
AIMS: Central diabetes insipidus (CDI), a typical complication caused by pituitary stalk injury, often occurs after surgery, trauma, or tumor compression around hypothalamic structures such as the pituitary stalk and optic chiasma. CDI is linked to decreased arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus, along with a deficit in circulating AVP and oxytocin. However, little has been elucidated about the changes in AVP neurons in CDI. Hence, our study was designed to understand the role of several pathophysiologic changes such as endoplasmic reticulum (ER) stress and apoptosis of AVP neurons in CDI. METHODS: In a novel pituitary stalk electric lesion (PEL) model to mimic CDI, immunofluorescence and immunoblotting were used to understand the underlying regulatory mechanisms. RESULTS: We reported that in CDI condition, generated by PEL, ER stress induced apoptosis of AVP neurons via activation of the PI3K/Akt and ERK pathways. Furthermore, application of N-acetylcysteine protected hypothalamic AVP neurons from ER stress-induced apoptosis through blocking the PI3K/Akt and ERK pathways. CONCLUSION: Our findings showed that AVP neurons underwent apoptosis induced by ER stress, and ER stress might play a vital role in CDI condition through the PI3K/Akt and ERK pathways.