RESUMO
The Nav1.1 voltage-gated sodium channel is a critical contributor to excitability in the brain, where pathological loss of function leads to such disorders as epilepsy, Alzheimer's disease, and autism. This voltage-gated sodium (Nav) channel subtype also plays an important role in mechanical pain signaling by primary afferent somatosensory neurons. Therefore, pharmacologic modulation of Nav1.1 represents a potential strategy for treating excitability disorders of the brain and periphery. Inactivation is a complex aspect of Nav channel gating and consists of fast and slow components, each of which may involve a contribution from one or more voltage-sensing domains. Here, we exploit the Hm1a spider toxin, a Nav1.1-selective modulator, to better understand the relationship between these temporally distinct modes of inactivation and ask whether they can be distinguished pharmacologically. We show that Hm1a inhibits the gating movement of the domain IV voltage sensor (VSDIV), hindering both fast and slow inactivation and leading to an increase in Nav1.1 availability during high-frequency stimulation. In contrast, ICA-121431, a small-molecule Nav1.1 inhibitor, accelerates a subsequent VSDIV gating transition to accelerate entry into the slow inactivated state, resulting in use-dependent block. Further evidence for functional coupling between fast and slow inactivation is provided by a Nav1.1 mutant in which fast inactivation removal has complex effects on slow inactivation. Taken together, our data substantiate the key role of VSDIV in Nav channel fast and slow inactivation and demonstrate that these gating processes are sequential and coupled through VSDIV. These findings provide insight into a pharmacophore on VSDIV through which modulation of inactivation gating can inhibit or facilitate Nav1.1 function.
Assuntos
Acetamidas/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Venenos de Aranha/farmacologia , Aranhas/química , Tiazóis/farmacologia , Animais , Humanos , Ativação do Canal Iônico/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Domínios Proteicos , Venenos de Aranha/química , Xenopus laevisRESUMO
BACKGROUND: Recent improvements in the survival of patients after esophagectomy have led to an increasing occurrence of gastric tube cancer (GTC). Removal of the reconstructed gastric tube, however, can lead to high morbidity and mortality. OBJECTIVE: To assess the feasibility and effectiveness of endoscopic submucosal dissection (ESD) for GTC. DESIGN: Retrospective study. SETTING: National Cancer Center Hospital, Tokyo, Japan. PATIENTS: We investigated patients with GTC after esophagectomy undergoing ESD from 1998 to 2011. INTERVENTION ESD MAIN OUTCOME MEASUREMENTS: Patient characteristics, endoscopic findings, technical results, histopathology including curability and Helicobacter pylori gastritis, and long-term outcomes. RESULTS: There were 51 consecutive patients with 79 lesions including 38 lesions (48%) meeting the absolute indication, 31 (39%) satisfying the expanded indications, and 10 (13%) falling outside such indications. The median procedure time was 90 minutes. There were 73 en bloc resections (92%), 59 en bloc resections with tumor-free margins (R0 resections, 75%), and 51 curative resections (65%) based on the Japanese Gastric Cancer Association criteria. Fifty patients (98%) were assessed as H pylori gastritis positive. Adverse events included 3 perforations (3.8%) during ESD and 2 delayed perforations (2.5%) without any emergency surgery and 3 delayed bleeding (3.8%). Local recurrence was detected in 4 patients (7.8%), and metachronous GTCs were identified in 18 patients (35%). Five patients (10%) died of GTC including 3 metachronous lesions. The 5-year overall survival rate was 68.4%, and the disease-specific survival rate was 86.7% with 100% for curative and 72.7% for non-curative patients during a median follow-up period of 3.8 years (range, 0-12.1 years). LIMITATION: Single-center retrospective study. CONCLUSIONS: ESD for GTC was feasible and effective for curative patients; however, long-term outcomes for non-curative patients were less satisfactory.
Assuntos
Dissecação/métodos , Esofagectomia/efeitos adversos , Mucosa Gástrica/cirurgia , Gastroscopia/métodos , Gastrostomia/efeitos adversos , Medição de Risco/métodos , Neoplasias Gástricas/cirurgia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Idoso , Intervalo Livre de Doença , Estudos de Viabilidade , Feminino , Seguimentos , Mucosa Gástrica/patologia , Gastrostomia/instrumentação , Humanos , Japão/epidemiologia , Masculino , Invasividade Neoplásica , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Estadiamento de Neoplasias , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Taxa de Sobrevida/tendências , Resultado do TratamentoRESUMO
Dravet syndrome is severe childhood-onset epilepsy, caused by loss of function mutations in the SCN1A gene, encoding for the voltage-gated sodium channel NaV1.1. The leading hypothesis is that Dravet is caused by selective reduction in the excitability of inhibitory neurons, due to hampered activity of NaV1.1 channels in these cells. However, these initial neuronal changes can lead to further network alterations. Here, focusing on the CA1 microcircuit in hippocampal brain slices of Dravet syndrome (DS, Scn1a A1783V/WT) and wild-type (WT) mice, we examined the functional response to the application of Hm1a, a specific NaV1.1 activator, in CA1 stratum-oriens (SO) interneurons and CA1 pyramidal excitatory neurons. DS SO interneurons demonstrated reduced firing and depolarized threshold for action potential (AP), indicating impaired activity. Nevertheless, Hm1a induced a similar AP threshold hyperpolarization in WT and DS interneurons. Conversely, a smaller effect of Hm1a was observed in CA1 pyramidal neurons of DS mice. In these excitatory cells, Hm1a application resulted in WT-specific AP threshold hyperpolarization and increased firing probability, with no effect on DS neurons. Additionally, when the firing of SO interneurons was triggered by CA3 stimulation and relayed via activation of CA1 excitatory neurons, the firing probability was similar in WT and DS interneurons, also featuring a comparable increase in the firing probability following Hm1a application. Interestingly, a similar functional response to Hm1a was observed in a second DS mouse model, harboring the nonsense Scn1a R613X mutation. Furthermore, we show homeostatic synaptic alterations in both CA1 pyramidal neurons and SO interneurons, consistent with reduced excitation and inhibition onto CA1 pyramidal neurons and increased release probability in the CA1-SO synapse. Together, these results suggest global neuronal alterations within the CA1 microcircuit extending beyond the direct impact of NaV1.1 dysfunction.
RESUMO
In this study, an empirical predictive model was developed based on the quantitative relationships between blend properties, critical quality attributes (CQA) and critical process parameters (CPP) related to blending and tableting. The blend uniformity and API concentration in the tablets were used to elucidate challenges related to the processability as well as the implementation of PAT tools. Thirty divergent ternary blends were evaluated on a continuous direct compression line (ConsiGma™ CDC-50). The trials showed a significant impact of the impeller configuration and impeller speed on the blending performance, whereas a limited impact of blend properties was observed. In contrast, blend properties played a significant role during compression, where changes in blend composition significantly altered the tablet quality. The observed correlations allowed to develop an empirical predictive model for the selection of process configurations based on the blend properties, reducing the number of trial runs needed to optimize a process and thus reducing development time and costs of new drug products. Furthermore, the trials elucidated several challenges related to blend properties that had a significant impact on PAT implementation and performance of the CDC-platform, highlighting the importance of further process development and optimization in order to solve the remaining challenges.
RESUMO
HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.