RESUMO
MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.
Assuntos
Melanoma , Receptor Tipo 1 de Melanocortina , Animais , Sistemas CRISPR-Cas , Humanos , Mamíferos/metabolismo , Melaninas/genética , Melanoma/genética , Melanoma/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , SuínosRESUMO
Oxidative stress is becoming increasingly implicated in the development of a variety of neurological disorders. However, the underlying mechanism remains elusive. In the present study, we investigated the function and related signal pathway which Cpg15, a neuronal-specific expressed neurotrophic factor, plays in the oxidative stress of neurons using a H2O2-treated N2a cell model. The results showed that the Cpg15 expression was decreased under oxidative stress, and overexpression of Cpg15 increased the activity of antioxidative SOD enzymes and decreased the expression level of prooxidative COX2 enzyme, and the level of oxidative products malondialdehyde (MDA), indicating its function and potential mechanism in alleviating the oxidative stress of cells. The results also indicated that the Nrf2/HO-1 antioxidative pathway was involved in the Cpg15-mediated alleviation of oxidative stress. Also, overexpression of Cpg15 activated the Nrf2 antioxidative pathway in the thalamus of the REM sleep-deprived mice. In conclusion, our results implied that supplemental expression of Cpg15 may alleviate oxidative stress in neuronal cells via regulating the redox enzymes or activating the Nrf2 antioxidant pathway.
Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Animais , Antioxidantes/metabolismo , Proteínas Ligadas por GPI , Peróxido de Hidrogênio , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Oxirredução , Estresse OxidativoRESUMO
OBJECTIVE: Exploring the role of amygdala enlargement (AE) in temporal lobe epilepsy (TLE) without ipsilateral mesial temporal sclerosis (MTS) using comprehensive presurgical workup tools including traditional tools, automatically volumetric analysis, high-density EEG (HD-EEG) source imaging (HD-ESI), and stereoelectroencephalography (SEEG). METHODS: Nine patients diagnosed with TLE-AE who underwent resective surgeries encompassing the amygdala were retrospectively studied. HD-ESI was obtained using 256-channel HD-EEG on the individualized head model. For automatic volumetric analysis, 48 matched controls were enrolled. Diagnosis and surgical strategies were based on a comprehensive workup following the anatomo-electro-clinical principle. RESULTS: At post-operative follow-up (average 30.9 months), eight patients had achieved Engel class I and one Engel class II recovery. HD-ESI yielded unifocal source estimates in anterior mesial temporal region in 85.7% of cases. Automatic volumetric analysis showed the AE sides were consistent with the values determined through other preoperative workup tools. Furthermore, the amygdala volume of the affected sides in AE was significantly greater than that of the larger sides in controls (p < 0.001). Meanwhile, the amygdala volume lateral index (LI) of AE was significantly higher than in controls (p < 0.001). SEEG analysis showed that ictal onsets arose from the enlarged amygdala (and hippocampus) in all cases. CONCLUSION: In addition to traditional workup tools, automatic volumetric analysis, HD-ESI on individualized head model, and invasive SEEG can provide evidence of epileptogenicity in TLE-AE. Resective surgical strategies encompassing the amygdala result in better prognosis. In suspected TLE cases, more attention should be focused on detecting enlargement of amygdala which sometimes is "hidden" in "MR-negative" non-MTS cases.
Assuntos
Epilepsia do Lobo Temporal , Tonsila do Cerebelo/diagnóstico por imagem , Tonsila do Cerebelo/cirurgia , Eletroencefalografia , Epilepsia do Lobo Temporal/diagnóstico por imagem , Epilepsia do Lobo Temporal/cirurgia , Hipocampo , Humanos , Imageamento por Ressonância Magnética , Estudos Retrospectivos , Lobo TemporalRESUMO
BACKGROUND: Autophagy is considered to be another restorative focus for the treatment of brain tumors. Although several research have demonstrated that melatonin induces autophagy in colon cancer and hepatoma cells, there has not been any direct evidence of whether melatonin is capable of inducing autophagy in human glioma cells. RESULTS: In the present research, we report that melatonin or its agonist, agomelatine, induced autophagy in A172 and U87-MG glioblastoma cells for a concentration-and time-dependent way, which was significantly attenuated by treatment with luzindole, a melatonin receptor antagonist. Furthermore, by suppressing autophagy at the late-stage with bafilomycin A1 and early stage with 3-MA, we found that the melatonin-induced autophagy was activated early, and the autophagic flux was complete. Melatonin treatment alone did not induce any apoptotic changes in the glioblastoma cells, as measured by flow cytometry. Western blot studies confirmed that melatonin alone prominently upregulated the levels of Beclin 1 and LC3 II, which was accompanied by an increase in the expression of Bcl-2, whereas it had no effect on the expression of Bax in the glioblastoma cells. Remarkably, co-treatment with 3-MA and melatonin significantly enhanced the apoptotic cell population in the glioblastoma cells, along with a prominent decrease in the expression of bcl-2 and increase in the Bax expression levels, which collectively indicated that the disruption of autophagy triggers the melatonin-induced apoptosis in glioblastoma cells. CONCLUSIONS: These results provide information indicating that melatonin may act as a common upstream signal between autophagy and apoptosis, which may lead to the development of new therapeutic strategies for glioma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Melatonina/farmacologia , Acetamidas/farmacologia , Apoptose/fisiologia , Autofagia/fisiologia , Neoplasias Encefálicas/fisiopatologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Glioblastoma/fisiopatologia , Humanos , Macrolídeos/farmacologia , Melatonina/agonistas , Receptores de Melatonina/agonistas , Receptores de Melatonina/antagonistas & inibidores , Triptaminas/farmacologiaRESUMO
The nanopore technique employs a nanoscale cavity to electrochemically confine individual molecules, achieving ultrasensitive single-molecule analysis based on evaluating the amplitude and duration of the ionic current. However, each nanopore sensing interface has its own intrinsic sensing ability, which does not always efficiently generate distinctive blockade currents for multiple analytes. Therefore, analytes that differ at only a single site often exhibit similar blockade currents or durations in nanopore experiments, which often produces serious overlap in the resulting statistical graphs. To improve the sensing ability of nanopores, herein we propose a novel shapelet-based machine learning approach to discriminate mixed analytes that exhibit nearly identical blockade current amplitudes and durations. DNA oligomers with a single-nucleotide difference, 5'-AAAA-3' and 5'-GAAA-3', are employed as model analytes that are difficult to identify in aerolysin nanopores at 100 mV. First, a set of the most informative and discriminative segments are learned from the time-series data set of blockade current signals using the learning time-series shapelets (LTS) algorithm. Then, the shapelet-transformed representation of the signals is obtained by calculating the minimum distance between the shapelets and the original signals. A simple logistic classifier is used to identify the two types of DNA oligomers in accordance with the corresponding shapelet-transformed representation. Finally, an evaluation is performed on the validation data set to show that our approach can achieve a high F1 score of 0.933. In comparison with the conventional statistical methods for the analysis of duration and residual current, the shapelet-transformed representation provides clearly discriminated distributions for multiple analytes. Taking advantage of the robust LTS algorithm, one could anticipate the real-time analysis of nanopore events for the direct identification and quantification of multiple biomolecules in a complex real sample (e.g., serum) without labels and time-consuming mutagenesis.
Assuntos
DNA/química , Nanoporos , Algoritmos , Toxinas Bacterianas/química , Sequência de Bases , Nucleotídeos/química , Proteínas Citotóxicas Formadoras de Poros/químicaRESUMO
It is reported that osteopontin has shown promising diagnostic value for malignant pleural mesothelioma (MPM), this meta-analysis aimed to establish the overall diagnostic accuracy of the osteopontin measurement for diagnosing MPM. Based on a systematic review of English language studies, the sensitivity, specificity and other measures of accuracy of osteopontin in the diagnosis of MPM were pooled using random-effects model. Summary receiver operating characteristic curves were used to summarize overall test performance. Seven publications met our inclusion criteria, the pooled sensitivity was 0.57 (95%CI: 0.52-0.61), specificity was 0.81, 95%CI: 0.79-0.84). The PLR was 3.78 (95%CI: 2.23-6.41), the NLR was 0.51 (95%CI: 0.38-0.67) and the DOR was 9.04 (95%CI: 5.28-15.48), the area under the summary receiver operating characteristic curve was 0.80. Our data suggest that osteopontin is likely to be a useful diagnostic marker for MPM, considering for the limited studies and patients included, larger studies are needed to confirm these findings.