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1.
J Anim Sci ; 98(3)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32191809

RESUMO

Hermetia illucens larvae meal (HILM) are rich in proteins and chitin, and represent an innovative feed ingredient for animals. However, little is known about the intestinal bacteria and immune homeostasis response of HILM as a fishmeal replacement on weanling piglets. Thus, this study aimed to investigate the changes in specific ileal and cecal bacterial populations and their metabolic profiles, and ileal immune indexes in weanling piglets fed with a diet containing HILM. A total of 128 weanling piglets were fed either a basal diet or 1 of 3 diets with 1%, 2%, and 4% HILM (HI0, HI1, HI2, and HI4, respectively). Each group consisted of 8 pens (replicates), with 4 pigs per pen. After 28 d of feeding, 8 barrows per treatment were euthanized, the ileal and cecal digesta, and ileal mucosa were collected for analyzing bacterial population and metabolic profiles, and immune indexes, respectively. Results showed that HILM increased (Pâ<â0.05, maximum in HI2) the number of Lactobacillus and Bifidobacterium in the ileum and cecum, but quadratically decreased (Pâ<â0.05, minimum in HI2) the number of Escherichia coli. In the cecum, the number of Firmicutes, Ruminococcus, Clostridium cluster IV, and Prevotella showed a quadratic response to increasing (Pâ<â0.05, maximum in HI2) HILM levels. Lactate and butyrate concentrations in the ileum and cecum were quadratically increased (Pâ<â0.05, maximum in HI2) with increasing HILM levels. In the cecum, the amines, phenol, and indole compounds concentrations were quadratically decreased (Pâ<â0.05, minimum in HI2) with increasing HILM levels, while total short-chain fatty acids and acetate concentrations were quadratically increased (Pâ<â0.05, maximum in HI2). In the ileum, the TLR4, NF-κB, MyD88, and TNF-α mRNA expressions were quadratically decreased (Pâ<â0.05, minimum in HI2) with increasing HILM levels, while the mRNA expression of IL-10, barrier function (MUC1, ZO-1, Occludin, and Claudin-2), and development-related genes (IGF-1, GLP-2, and EGF) was quadratically increased (Pâ<â0.05, maximum in HI2). Furthermore, the changes in the mucosal gene expression were associated with changes in the bacterial populations and their metabolites. Collectively, these results showed that a diet supplemented with 2% HILM affected specific bacterial populations and metabolic profiles, and maintained ileal immune status. These findings provide new insights into the use of insect meal as a suitable alternative protein source for swine feeding.

2.
Plant Dis ; : PDIS08191795RE, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32065566

RESUMO

Production of pycnidia and pycnidiospores by Macrophomina phaseolina is not often seen in vitro. The objective of this study is to develop a simple and effective technique to obtain pycnidiospores of M. phaseolina isolates in vitro and to evaluate germination rates and pathogenicity of pycnidiospores. We found M. phaseolina isolates can produce pycnidia on oatmeal agar (OMA) under ultraviolet light with 365 nm wavelength (UV). For evaluating the effect of OMA and UV on growth of M. phaseolina, combinations of two agar media and three lighting conditions were tested. The results confirm that all six M. phaseolina isolates produced pycnidia only on OMA under UV. The pycnidiospores produced on OMA under UV had germination rates higher than 90%. In pathogenicity tests, inoculation with the pycnidiospores showed symptoms later than inoculation with hypha-colonized toothpicks. Significant differences in the pathogenicity is detected between isolates Mp2014003 and Mp2014024 when inoculation is done with the pycnidiospores (P < 0.001), but not when hypha-colonized toothpicks are used as inoculum (P = 0.091). This study provides a new method for obtaining pycnidiospores of M. phaseolina for future investigations.

3.
Mol Plant Pathol ; 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32105402

RESUMO

SsITL, a secretory protein of the necrotrophic phytopathogen Sclerotinia sclerotiorum, was previously reported to suppress host immunity at the early stages of infection. However, the molecular mechanism that SsITL uses to inhibit plant defence against S. sclerotiorum has not yet been elucidated. Here, we report that SsITL interacted with a chloroplast-localized calcium-sensing receptor, CAS, in chloroplasts. We found that CAS is a positive regulator of the salicylic acid signalling pathway in plant immunity to S. sclerotiorum and CAS-mediated resistance against S. sclerotiorum depends on Ca2+ signalling. Furthermore, we showed that SsITL could interfere with the plant salicylic acid (SA) signalling pathway and SsITL-expressing transgenic plants were more susceptible to S. sclerotiorum. However, truncated SsITLs (SsITL-NT1 or SsITL-CT1) that lost the ability to interact with CAS do not affect plant resistance to S. sclerotiorum. Taken together, our findings reveal that SsITL inhibits SA accumulation during the early stage of infection by interacting with CAS and then facilitating the infection by S. sclerotiorum.

4.
Mar Pollut Bull ; 151: 110810, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32056603

RESUMO

The distribution of 77 antibiotics in the coastal water and sediment from 3 bays of the East China Sea was investigated. There were 43 and 25 antibiotics detected with total concentrations of 30.8-2106.1 ng/L and 2.2-99.9 ng/g in water and sediment, respectively. Approximately 83.0% and 85.4% of the individual antibiotic concentrations were lower than 5.0 ng/L in water and 1.0 ng/g in sediment. Clindamycin (1.2-1507.9 ng/L, mean 183.8 ng/L) and erythromycin (ND-45.2 ng/g, mean 3.4 ng/g) were the most abundant in water and sediment, respectively. Ecological risk assessment revealed that the joint toxicity was enhanced when multiple antibiotics were present simultaneously. A decrease in the total antibiotic concentration and the ecological risk in water was observed from nearshore to offshore. Three antibiotics (sulfamethoxypyridazine, sulfamethoxazole and cinoxacin) were selected to be prioritized based on ecological risks for antibiotics monitoring and management of the coastal water in the East China Sea.

Assuntos
Antibacterianos/análise , Monitoramento Ambiental , Poluentes Químicos da Água/análise , China , Sedimentos Geológicos , Medição de Risco
5.
Anal Chem ; 92(6): 4512-4517, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083471

RESUMO

A compact isotope ratio sensor based on laser absorption spectroscopy at 2.7 µm was developed for high precision and simultaneous measurements of the D/H, 18O/16O and 17O/16O isotope ratios in glacier water. Measurements of the oxygen and hydrogen isotope ratios in glacier water demonstrate a 1σ precision of 0.3 for Î´18O, 0.2 for Î´17O, and 0.5 for Î´2H, respectively. The Î´ values of the working standard glacier water obtained by the calibrated sensor system is basically identical to the IRMS measurement results with a very high calibration accuracy from 0.17 to 0.75. Preliminary results on the reproducibility measurements display a standard deviation of 0.13 for Î´18O, 0.13 for Î´17O, and 0.64 for Î´2H, respectively.

6.
J Pharm Pharmacol ; 72(4): 496-506, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31975457

RESUMO

OBJECTIVES: Zein nanoparticles (Zein NPs) were used as a hydroxyapatite (HA) biomineralization template to generate HA/Zein NPs. Doxorubicin hydrochloride (DOX) was loaded on HA/Zein NPs (HA/Zein-DOX NPs) to improve its pH-sensitive release, bioavailability and decrease cardiotoxicity. METHODS: HA/Zein-DOX NPs were prepared by phase separation and biomimetic mineralization method. Particle size, polydispersity index (PDI), Zeta potential, transmission electron microscope, X-ray diffraction and Fourier-transform infrared spectroscopy of HA/Zein-DOX NPs were characterized. The nanoparticles were then evaluated in vitro and in vivo. KEY FINDINGS: The small PDI and high Zeta potential demonstrated that HA/Zein-DOX NPs were a stable and homogeneous dispersed system and that HA was mineralized on Zein-DOX NPs. HA/Zein-DOX NPs showed pH-sensitive release. Compared with free DOX, HA/Zein-DOX NPs increased cellular uptake which caused 7 times higher in-vitro cytotoxicity in 4T1 cells. Pharmacokinetic experiments indicated the t1/2ß and AUC0- t of HA/Zein-DOX NPs were 2.73- and 3.12-fold higher than those of DOX solution, respectively. Tissue distribution exhibited HA/Zein-DOX NPs reduced heart toxicity with lower heart targeting efficiency (18.58%) than that of DOX solution (37.62%). CONCLUSION: In this study, HA/Zein-DOX NPs represented an antitumour drug delivery system for DOX in clinical tumour therapy with improved bioavailability and decreased cardiotoxicity.

7.
Sci Total Environ ; 710: 136199, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-31918186

RESUMO

Nowadays, as microgrid development has been limited by the high cost of its energy storage system (ESS), many relevant literatures on applied energy have emphasized the role of ESS incentive policies for microgrid. However, since energy storage technology in microgrid is complex, it still remains an issue of high difficulty to derive the optimal ESS incentives for microgrid development. In this study, a real option game model, which combines evolutionary game theory with real options, is developed to explore the socially optimal ESS incentive policies for microgrid by utilizing the fluctuation amplitudes and equilibrium positions of microgrid development under different ESS policy options, that include the initial cost subsidy, the feed-in tariffs (FIT), and the production tax credit. Moreover, we provide a numerical example to simulate and compare the fluctuation results of microgrid development under different ESS incentive policies. The results indicate that the FIT has the greatest effect on microgrid development. However, regarding the limitations of government's incentive mechanisms, its combination with other ESS incentive policies, e.g., initial cost subsidy, and tax credit, is shown to be more effective for microgrid development than the FIT only. In addition, appropriately extending the implementation period of FIT can also help to overcome the limitations of FIT mechanism so as to effectively facilitate the development of microgrid.

8.
Acta Pharm ; 70(2): 227-237, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31955141

RESUMO

Curcumin is a lipophilic anti-cancer compound extracted from turmeric. Our previous study demonstrated that the curcumin-loaded nanostructured lipid carrier (Cur-NLC) exhibits superior anti-cancer activity in inhibiting proliferation as well as inducing apoptosis of human HepG2 cells compared to native curcumin. This study aims to unveil the mechanisms underlying the pro-apoptotic effect of Cur-NLC on HepG2 cells. Evidence indicates that low expression of death receptors (DRs) on cancer cell membranes leads to attenuated apoptosis signaling. This study showed that Cur-NLC significantly increased total expression of DR5 protein while simultaneously upregulated cell membrane expression of DR5. Cur-NLC significantly increased caspase-8 and caspase-3 activities, accompanied by increased apoptosis. Furthermore, enhanced apoptosis was inhibited in the presence of a pan-caspase inhibitor, Z-VAD-FMK. Therefore, Cur-NLC induced activation of the extrinsic apoptosis pathway via modulating the DR5/caspase-8/-3 mediated apoptosis pathway in HepG2 cells, suggesting that Cur-NLC is a promising therapeutic agent or supplement for the treatment of hepatocellular carcinoma.

9.
Anal Chem ; 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31984730

RESUMO

The total OH reactivity (kOH') is an important parameter for quantitative assessment of the atmospheric oxidation capacity. Although laboratory measurement of kOH' has been achieved 20 years ago, the instruments required are often costly and complex. Long-term atmospheric observations remain challenging and elusive. In this work, a novel instrument combining laser-flash photolysis with a mid-infrared Faraday rotation spectrometer (LFP-FRS) has been developed for the measurement of kOH' and for studying gas phase free radical kinetics. The reactor is composed of a Herriott-type optical multipass cell, and OH radicals were generated by flash photolysis of ozone with a 266 nm pulsed Nd:YAG laser. The decay of the OH signal was directly measured with a time-resolved FRS spectrometer at 2.8 µm. The overlapping path length between the pump beam and probe beam was 25 m. High performance was achieved by subtracting the signals before and after flash photolysis to eliminate interferences caused by H2O absorption and background drift. The optimum precisions (1σ) of OH concentration and kOH' measurement were 4 × 106 molecules cm-3 and 0.09 s-1 over data acquisition times of 56 and 112 s, respectively. The performance of the system was evaluated by the reaction of OH with CO and NO. The measured rate coefficients (kOH+CO and kOH+NO) were in good agreement with values reported in the literature. The developed LFP-FRS provides a new, high precision, and highly selective tool for atmospheric chemistry research of OH radicals and other transient paramagnetic free radicals such as HO2 radicals.

10.
Cells ; 9(1)2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940841

RESUMO

Activation of hepatic macrophages represents the critical driving force to promote cholestatic liver injury. Exosomes, as important small extracellular vesicles released by almost all types of cells, contribute to intercellular communication. We previously reported that cholangiocyte-derived exosomal long noncoding RNA (lncRNA) H19 plays a vital role in disrupting bile acid homeostasis in hepatocytes and promoting the activation of hepatic stellate cells (HSCs). Exosomal H19 derived from cholangiocytes was rapidly taken up by Kupffer cells. However, the mechanistic links between exosomal lncRNA H19 and macrophage-driven inflammation in cholestasis remain unclear. Here, we reported that the hepatic H19 level was closely correlated with macrophage activation and hepatic fibrosis in both Mdr2-/- and bile duct ligation (BDL) cholestatic mouse models, as well as in human primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients. Exosomal H19 significantly induced the expression and secretion of chemokine (C-C motif) ligand 2 (CCL-2) and interleukin 6 (IL-6) in Kupffer cells. H19-enriched exosomes enhanced the activation M1 polarization of Kupffer cells and promoted the recruitment and differentiation of bone marrow-derived macrophages, which were inhibited by a CCL-2 pharmacological inhibitor. In conclusion, Cholangiocyte-derived exosomal H19 played a critical role in macrophage activation, differentiation, and chemotaxis through CCL-2/CCR-2 signaling pathways, which represent a therapeutic target for cholestatic liver diseases.

11.
Ren Fail ; 42(1): 122-130, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31957527

RESUMO

Objective: To evaluate the clinical efficacy and safety of leflunomide (LEF) combined with prednisone for the treatment of PLA2R-associated primary membranous nephropathy (PMN) and changes in anti-PLA2R antibody titers after treatment.Methods: Sixty patients with nephrotic syndrome, biopsy-proven MN and anti-PLA2R antibody positivity were included in this study conducted from December 2017 to February 2019. The patients were randomly divided into an experimental group (n = 30) and a control group (n = 30). The patients in the experimental group were treated with LEF combined with prednisone, whereas the patients in the control group were treated with cyclophosphamide (CTX) combined with prednisone. We assessed 24-h urinary protein and serum albumin levels, kidney function markers, blood lipid levels and anti-PLA2R antibody titers before and after treatment. Adverse reactions during treatment were recorded.Results: After 16 weeks of treatment, there were 2 cases of complete remission and 6 cases of partial remission in the experimental group, with a total effective rate of 26.67%. In the control group, there were 4 cases of complete remission and 8 cases of partial remission, with a total effective rate of 40% (p > .05). After 24 weeks of treatment, the total effective rates of the experimental and control groups were 66.67% and 76.67%, respectively (p > .05). There were no significant differences in 24-h urinary protein, serum albumin, kidney function marker or blood lipid levels between the two groups after treatment (p > .05). However, there were fewer adverse reactions in the experimental group than in the control group (p < .05). After treatment, serum anti-PLA2R antibody titers were clearly decreased in patients with complete remission and partial remission (p < .05), but these levels remained relatively high in patients without remission (p > .05).Conclusion: LEF combined with prednisone has a certain efficacy for the treatment of PLA2R-associated PMN and provokes few adverse reactions. A large-sample randomized double-blind controlled study with a long follow-up period is needed to verify the efficacy of LEF combined with prednisone.

12.
Am J Pathol ; 190(2): 469-483, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31783009

RESUMO

Liver cancer is the third leading cause of cancer-related death worldwide. Herein, we show that miR-149* serves as a novel tumor suppressor for liver tumorigenesis. Mice with genetic deletion of miR-149* (miR-149*-/- mice), which caused loss of both miR-149 and miR-149*, were considerably more susceptible to acute liver injury and hepatic carcinogenesis induced by diethylnitrosamine than wild-type mice, accompanied by increased compensatory proliferation and up-regulated gene expression of certain inflammatory cytokines. miR-149* mimics dramatically impaired liver cancer cell proliferation and migration in vitro and blocked liver cancer progression in a xenograft model. Furthermore, miR-149* strongly suppressed NF-κB signaling and repressed tumor necrosis factor receptor type 1-associated death domain protein expression in the NF-κB signaling pathway. These results reveal that miR-149*, as a novel liver tumor suppressor, may serve as a potential therapeutic target for liver cancer treatment.

13.
Appl Opt ; 58(32): 8743-8750, 2019 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-31873651

RESUMO

We report the development of an improved spherical mirror multipass-cell-based interband cascade laser (ICL) spectrometer for ambient formaldehyde (HCHO) detection. The multipass cell consists of two easily manufactured spherical mirrors that are low cost, and have a simple structure, large mirror area utilization, and dense spot pattern. Optical interference caused by the multipath cell was largely reduced, resulting in good sensitivity. Using wavelength modulation spectroscopy (WMS), a detection precision (${1} \sigma$1σ) of 51 pptv in 10 s was achieved with an absorption pathlength of 96 m, which compared favorably with the performance of other state-of-the-art instruments. The precision can be further improved by using a long absorption pathlength configuration and by removing fringe-like optical noise caused by the collimation lens. Ambient application of the developed spectrometer was demonstrated.

14.
Zhongguo Zhong Yao Za Zhi ; 44(20): 4538-4544, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31872643

RESUMO

Through using the theoretical research results of traditional Chinese medicine resource management and sustainable utilization as references,this article systematically analyzes the relationship between economic subsystems for utilization of wild renewable resources in Tibetan medicine,resources,environment and social systems. It sorts out and designs the relationship diagram of the sustainable utilization system for Tibetan medicine resources,input and output relationship diagrams of economics subsystems for Tibetan medicine resources,development model diagram for Tibetan medical industrial chain,market development flow chart of wild herb resources of Tibetan medicine,causal circuit diagram of sustainable use mechanism for Tibetan medicine resources,cause tree of the stock and function for Tibetan medicine resources,cause tree of total income for Tibetan medicine resource utilization and tree diagram of compensation fund usage of Tibetan medicine resources. It provides an innovative research method for Tibetan medicine resources,and theoretical basis and relevant countermeasure for constructing sustainable approaches to development of wild renewable resources in Tibetan medicine.

Assuntos
Medicina Tradicional Tibetana , Plantas Medicinais , Medicina Tradicional Chinesa , Desenvolvimento Sustentável , Tibet
15.
Opt Express ; 27(21): 30517-30529, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31684298

RESUMO

Injecting radio frequency (RF) white noise to the current driving of the laser can broaden the laser emission linewidth and efficiently suppress cavity-mode noise in off-axis integrated cavity output spectroscopy (OA-ICOS). The effect of the injected RF noise level on the cavity-mode noise and the deformation of the absorption line shape in off-axis integrated cavity output spectroscopy (OA-ICOS) with a distributed feedback laser (DFB) at 1.65 µm were investigated. We measured methane at different concentrations between 0.1 ppmv and 2 ppmv associated with a -20 dBm RF noise injection. A linear spectral response of the intensity of the cavity output spectra with the CH4 concentration was observed. A threefold improvement in the detection limit was achieved compared to unperturbed OA-ICOS. The response time of the improved OA-ICOS system is about 30 s and the minimum detectable concentration (MDC) of CH4 is 7.6 ppbv, which corresponds to a minimum detectable fractional absorption scaled to the path length of 7.3 × 10-10 cm-1. The noise equivalent absorption sensitivity of the system is 5.51 × 10-9 cm-1Hz-1/2.

16.
Toxins (Basel) ; 11(11)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671808

RESUMO

Phytotoxins, secondary metabolites toxic to plants and produced by fungi, are believed to play an important role in disease development by targeting host cellular machineries and/or interfering with host immune responses. The Ascochyta blight diseases on different legume plants are caused by Ascochyta and related taxa, such as Phoma. The causal agents of the Ascochyta blight are often associated with specific legume plants, showing a relatively narrow host range. The legume-associated Ascochyta and Phoma are known to produce a diverse array of polyketide-derived secondary metabolites, many of which exhibited significant phytotoxicity and have been claimed as virulence or pathogenicity factors. In this article, we reviewed the current state of knowledge on the diversity and biological activities of the phytotoxic compounds produced by Ascochyta and Phoma species. Also, we touched on the secondary metabolite biosynthesis gene clusters identified thus far and discussed the role of metabolites in the fungal biology.

17.
Microorganisms ; 7(11)2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671828

RESUMO

Trichoderma species are a group of fungi which is widely distributed in major terrestrial ecosystems; they are also commonly used as biocontrol agents for many plant diseases. A virus, namely Trichoderma harzianum hypovirus 1 (ThHV1), was identified in T. harzianum isolate T-70, and also infected isolate T-70D, together with its defective RNA (ThHV1-S). The ThHV1 genome possessed two Open Reading Frames (ORFs), namely ORF1 and ORF2. The start codon of ORF2 overlapped with the stop codon of ORF1 in a 43 nt long region. The polypeptide encoded by ORF2 of ThHV1 shared sequence similarities with those of betahypoviruses, indicating that ThHV1 is a novel member of Hypoviridea. Isolate T-70D, carrying both ThHV1 and ThHV1-S, showed abnormal biological properties, notably a decreased mycoparasitism ability when compared with isolate T-70. Both ThHV1 and ThHV1-S could be vertically transmitted to conidia and horizontally transmitted to T. harzianum isolate T-68 and T. koningiopsis T-51. The derivative strains carrying both ThHV1 and ThHV1-S showed decreased mycoparasitism ability, whereas strains carrying ThHV1 alone were normal, indicating that ThHV1-S is closely associated with the decreased mycoparasitism ability of T. harzianum isolate T-70D. ThHV1 was widely detected in isolates of T. harzianum, T. koningiopsis and T. atroviride originating from soil of China. Therefore, viruses in fungal biocontrol agents may also be a factor associated with the stability of their application.

18.
Clin Chem Lab Med ; 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31714884
19.
Opt Lett ; 44(23): 5659-5662, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31774747

RESUMO

We report on a narrowband, nonresonant periodically poled lithium niobate (PPLN) optical parametric oscillator using a volume Bragg grating (VBG) as the spectral narrowing element. Pumping by a Nd:YVO4 laser at 1.06 µm, a maximum output power of 4.75 W is achieved at a repetition rate of 20 kHz for a conversion efficiency of 47.5%. Both signal and idler spectra are narrowed to less than 2 nm, at good beam quality and stability.

20.
Adv Exp Med Biol ; 1101: 67-89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31729672

RESUMO

Because of high spatial-temporal resolution of neural signals obtained by invasive recording, the invasive brain-machine interfaces (BMI) have achieved great progress in the past two decades. With success in animal research, BMI technology is transferring to clinical trials for helping paralyzed people to restore their lost motor functions. This chapter gives a brief review of BMI development from animal experiments to human clinical studies in the following aspects: (1) BMIs based on rodent animals; (2) BMI based on non-human primates; and (3) pilot BMIs studies in clinical trials. In the end, the chapter concludes with a summary of potential opportunities and future challenges in BMI technology.

Assuntos
Interfaces Cérebro-Computador , Animais , Interfaces Cérebro-Computador/normas , Interfaces Cérebro-Computador/tendências , Ensaios Clínicos como Assunto , Humanos