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Background: Influenza virus is a respiratory pathogen, which causes high degree of mortality and morbidity during seasonal epidemics and sporadic pandemics. By selecting conserved antigenic proteins, for example, hemagglutinin small subunit (HA2) and nucleoprotein (NP), we aimed to develop a vaccine based on a fusion protein leading to both cellular and humoral responses that are the most challenging aspects in designing a universal vaccine. Materials and Methods: The bioinformatics analysis was performed for HA2-NP structure and function prediction. Primers for the antigenic part of NP were designed using bioinformatics tools. The desired product was amplified via polymerase chain reaction using the designed primers, which was then penetrated into T vector, followed by insertion into pET28a vector in order to construct pET28a/NP. The pET28a/HA2, previously generated in our lab, was digested with the same restriction enzymes as pET28a/NP (HindIII/Xhol). Then, NP was inserted to the downstream region of HA2 to construct pET28a/HA2. Results: The generated pET28a/HA2-NP was transformed into Escherichia coli BL21 (DE3). The expression was induced by isopropyl ß-d-l-thiogalactopyranoside. The results showed that the antigenic segment of NP was successfully cloned into pET28a/ HA2. The protein band of HA2-NP was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirmed by Western blotting and purified with Ni-NTA purification system (QIAGEN, Germany). Conclusion: As currently available vaccines can cause some allergic reactions, using a chimer protein based on the bioinformatics analysis is continual, safe, and affordable, thus stimulating both cellular and humoral immunity systems. Our construct could potentially provide a basis for a universal vaccine candidate.
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The immunogenicity and protective properties of the designed recombinant fusion peptide of 3M2e and truncated nucleoprotein (trNP), originating from Influenza A virus, were investigated in the BALB/c mice model in comparison with the Mix protein (3M2e + trNP). The results were evaluated by antibody response, cytokine production, lymphocyte proliferation assay, and mortality rate after challenge with homologous (H1N1) and heterologous (H3N2) influenza viruses in BALB/c mice. The animals that received the chimer protein with or without adjuvant had more specific antibody responses and elicited memory CD4 T cells, and cytokines of Th1 and Th2 cells compared to the Mix protein. Moreover, the Mix protein, like the recombinant chimer protein, provided equal and effective protection against both homologous and heterologous challenges in mice. Nevertheless, the chimer protein demonstrated superior immune protection compared to the Mix protein. The percentage of survived animals in the adjuvanted protein group (78.4%) was less than the non-adjuvanted one (85.7%). However, the Mix protein plus Alum could induce protective immunity in only 57.1% and 42.8% of homologous and heterologous virus-challenged mice, respectively. Regarding the sufficient immunogenicity and protectivity of the chimer protein construct against influenza viruses, the findings of the study suggest that the chimer protein without a requirement of adjuvant can be used as an adequate vaccine formulation to protect against a broad spectrum of influenza viruses.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Animales , Ratones , Humanos , Subtipo H3N2 del Virus de la Influenza A , Anticuerpos Antivirales , Adyuvantes Inmunológicos/farmacología , Ratones Endogámicos BALB CRESUMEN
PURPOSE: Influenza is one of the most important agents of pandemic outbreak causing substantial morbidity and mortality. Vaccination strategies of influenza must be adapted annually due to constant antigenic changes in various strains. Therefore, the present study was conducted to evaluate protective immunity of the conserved influenza proteins. METHODS: For this purpose, three tandem repeats of M2e (3M2e) and NP were separately expressed in E. coli and were purified using column chromatography. Female Balb/c mice were injected intradermally with a combination of the purified 3M2e and NP alone or formulated with Alum (AlOH3) adjuvant in three doses. The mice were challenged by intranasal administration of H1N1 (A/PR/8/34) 2 weeks after the last vaccination. RESULTS: The results demonstrated that recombinant NP and M2e proteins are immunogenic and could efficiently elicit immune responses in mice compared to non-immunized mice. The combination of 3M2e and NP supplemented with Alum stimulated both NP and M2e-specific antibodies, which were higher than those stimulated by each single antigen plus Alum. In addition, the secretion of IFN-γ and IL-4 as well as the induction of lymphocyte proliferation in mice received the mixture of these proteins with Alum was considerably higher than other groups. Moreover, the highest survival rate (86%) with the least body weight change was observed in the mice immunized with 3M2e and NP supplemented with Alum followed by the mice received NP supplemented with Alum (71%). CONCLUSION: Accordingly, this regimen can be considered as an attractive candidate for global vaccination against influenza.
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Compuestos de Alumbre/química , Vacunas contra la Influenza , Proteínas de la Nucleocápside , Proteínas Recombinantes , Proteínas de la Matriz Viral , Adyuvantes Inmunológicos/química , Animales , Anticuerpos Antivirales/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/genética , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunación , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunologíaRESUMEN
OBJECTIVES: Vaccination is the most effective preventive strategy for influenza disease. As the virus undergoes high antigenic drift, it requires a constant reformulation to obtain high protection. RESULTS: Immunogenicity of a purified chimeric protein containing conserved regions of influenza A/H1N1 viruses including the Hemagglutinin stalk domain, Nucleoprotein, and Matrix protein produced in a prokaryotic system was assessed in vitro and in vivo, alone or in combination with adjuvants by evaluating antibody responses, cytokine production, lymphocyte proliferative assay, and mortality rate after challenge. The animals that received the chimeric protein had specific antibody responses, elicited memory CD4 cells, cytokines of Th1 and Th2 cells and showed 75% protection against influenza virus lethal challenge. The animals injected with the chimeric protein supplemented with Alum showed improved immune responses, but they had 67% protection. In other words, although Alum adjuvant enriched the chimera specific immune responses potently, it could not enhance its protectivity. CONCLUSION: Regarding the immunogenicity and protectivity of the chimeric protein construct against influenza, findings of the study suggested that the chimeric protein could be considered as a promising influenza vaccine candidate.
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Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes de Fusión , Vacunas de Subunidad/inmunología , Animales , Modelos Animales de Enfermedad , Perros , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Vacunas contra la Influenza/administración & dosificación , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/genética , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas de Subunidad/administración & dosificaciónRESUMEN
PURPOSE: Autophagy plays a key role in host defence responses against microbial infections by promoting degradation of pathogens and participating in acquired immunity. The interaction between autophagy and viruses is complex, and this pathway is hijacked by several viruses. Influenza virus (IV) interferes with autophagy through its replication and increases the accumulation of autophagosomes by blocking lysosome fusion. Thus, autophagy could be an effective area for antiviral research. METHODOLOGY: In this study, we evaluated the effect of autophagy on IV replication. Two cell lines were transfected with Beclin-1 expression plasmid before (prophylactic approach) and after (therapeutic approach) IV inoculation.Results/Key findings. Beclin-1 overexpression in the cells infected by virus induced autophagy to 26â%. The log10haemagglutinin titre and TCID50 (tissue culture infective dose giving 50â% infection) of replicating virus were measured at 24 and 48 h post-infection. In the prophylactic approach, the virus titre was enhanced significantly at 24 h post-infection (P≤0.01), but it was not significantly different from the control at 48 h post-infection. In contrast, the therapeutic approach of autophagy induction inhibited the virus replication at 24 and 48 h post-infection. Additionally, we showed that inhibition of autophagy using 3-methyladenine reduced viral replication. CONCLUSION: This study revealed that the virus (H1N1) titre was controlled in a time-dependent manner following autophagy induction in host cells. Manipulation of autophagy during the IV life cycle can be targeted both for antiviral aims and for increasing viral yield for virus production.
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Autofagia/inmunología , Beclina-1/metabolismo , Virus de la Influenza A/crecimiento & desarrollo , Infecciones por Orthomyxoviridae/inmunología , Replicación Viral/inmunología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia/efectos de los fármacos , Beclina-1/genética , Perros , Hemaglutininas/inmunología , Virus de la Influenza A/genética , Células de Riñón Canino Madin Darby , Infecciones por Orthomyxoviridae/virología , Transfección/métodos , Carga ViralRESUMEN
OBJECTIVES: To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome. RESULTS: A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome. CONCLUSIONS: Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.
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Estomatitis Vesicular/metabolismo , Proteínas Virales/metabolismo , Virosomas/metabolismo , Técnicas de Transferencia de Gen , Transfección , Proteínas Virales/genética , Virosomas/genéticaRESUMEN
OBJECTIVES: To evaluate MDCK and MDCK-SIAT1 cell lines for their ability to produce the yield of influenza virus in different Multiplicities of Infection. RESULTS: Yields obtained for influenza virus H1N1 grown in MDCK-SIAT1 cell was almost the same as MDCK; however, H3N2 virus grown in MDCK-SIAT1 had lower viral titers in comparison with MDCK cells. The optimized MOIs to infect the cells on plates and microcarrier were selected 0.01 and 0.1 for H1N1 and 0.001 and 0.01 for H3N2, respectively. CONCLUSIONS: MDCK-SIAT1 cells may be considered as an alternative mean to manufacture cell-based flu vaccine, especially for the human strains (H1N1), due to its antigenic stability and high titer of influenza virus production.
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Técnicas de Cultivo de Célula , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H3N2 del Virus de la Influenza A/crecimiento & desarrollo , Células de Riñón Canino Madin Darby/citología , Células de Riñón Canino Madin Darby/virología , Animales , Adhesión Celular , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Pollos , Dextranos , Perros , Pruebas de Hemaglutinación/métodos , Hemaglutinación por Virus , Vacunas ViralesRESUMEN
When reaching to grasp, we coordinate how we preshape the hand with how we move it. To ask how motor cortical neurons participate in this coordination, we examined the interactions between reach- and grasp-related neuronal ensembles while monkeys reached to grasp a variety of different objects in different locations. By describing the dynamics of these two ensembles as trajectories in a low-dimensional state space, we examined their coupling in time. We found evidence for temporal compensation across many different reach-to-grasp conditions such that if one neural trajectory led in time the other tended to catch up, reducing the asynchrony between the trajectories. Granger causality revealed bidirectional interactions between reach and grasp neural trajectories beyond that which could be attributed to the joint kinematics that were consistently stronger in the grasp-to-reach direction. Characterizing cortical coordination dynamics provides a new framework for understanding the functional interactions between neural populations.
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Fuerza de la Mano , Destreza Motora , Neuronas/fisiología , Animales , Fenómenos Biomecánicos , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Femenino , Mano/fisiología , Macaca mulattaRESUMEN
INTRODUCTION: An unusual incidence of tuberculosis in different parts of the body is called tuberculomas. The rate of brain tuberculosis is rare. CASE PRESENTATION: The following case of tuberculamas of the brain, presented by enhancing rings of meninges, is reported because of its rarity. It was a case of brain tuberculomas in a 15-year-old girl with primary symptoms of headache and general weakness, and no signs of primary pulmonary infection. DISCUSSION: The subject underwent computerized tomography (CT) and magnetic resonance imaging (MRI) of the brain. Microbiological tests (acid fast bacilli smear-AFB, and culture of biopsy specimen) were applied subsequently. According to the results, the problem was diagnosed as brain tuberculomas. After operation she was completely treated with anti-TB drugs. Although brain tuberculosis is rare, it was diagnosed on the basis of histopathology and the patient's successful response to anti-tuberculous drug treatment.
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Though the joints of the arm and hand together comprise 27 degrees of freedom, an ethological movement like reaching and grasping coordinates many of these joints so as to operate in a reduced dimensional space. We used a generalized linear model to predict single neuron responses in primary motor cortex (MI) during a reach-to-grasp task based on 40 features that represent positions and velocities of the arm and hand in joint angle and Cartesian coordinates as well as the neurons' own spiking history. Two rhesus monkeys were trained to reach and grasp one of five objects, located at one of seven locations while we used an infrared camera motion-tracking system to track markers placed on their upper limb and recorded single-unit activity from a microelectrode array implanted in MI. The kinematic trajectories that described hand shaping and transport to the object depended on both the type of object and its location. Modeling the kinematics as temporally extensive trajectories consistently yielded significantly higher predictive power in most neurons. Furthermore, a model that included all feature trajectories yielded more predictive power than one that included any single feature trajectory in isolation, and neurons tended to encode feature velocities over positions. The predictive power of a majority of neurons reached a plateau for a model that included only the first five principal components of all the features' trajectories, suggesting that MI has evolved or adapted to encode the natural kinematic covariations associated with prehension described by a limited set of kinematic synergies.
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Potenciales de Acción/fisiología , Fuerza de la Mano/fisiología , Corteza Motora/fisiología , Desempeño Psicomotor/fisiología , Animales , Femenino , Macaca mulattaRESUMEN
BACKGROUND: The hemagglutinin molecule of influenza virus is considered as an ideal model to study biological processes as well as the effect of glycosylation on the function of glycoproteins. OBJECTIVES: The large subunit of the influenza virus A/New Caledonia/20/99 (H1N1) hemagglutinin (HA1) was expressed in recombinant Escherichia coli containing the glycosylation system of Campylobacter jejuni. This viral glycoprotein contains glycosylation motifs recognized by prokaryotic and eukaryotic oligosaccharyltransferases. METHODS: In order to express the hemagglutinin large subunit gene, the gene was amplified using reverse transcription polymerase chain reaction (RT-PCR), and it was cloned in pET22b for periplasmic expression. RESULTS: Western blotting and lectin blotting bands confirmed glycosylation of the HA1 in recombinant E. coli. CONCLUSION: Such a successful accomplishment of hemagglutinin expression in recombinant E. coli can be used to construct carbohydrates in hemagglutinin molecules of different strains in order to produce effective antigens for vaccine and rapid diagnostic kits against new emerging viruses.
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Escherichia coli/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Procesamiento Proteico-Postraduccional , Campylobacter jejuni/genética , Escherichia coli/genética , Glicosilación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Redes y Vías Metabólicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Current influenza virus vaccines provide protection in part by antibodies induced to the two surface glycoproteins, the hemagglutinin and the neuraminidase. As a result of the continuous antigenic drift of these glycoproteins, a frequent update of the composition of influenza vaccines is required. The search for more conserved viral epitopes which would induce protective immunity against seasonal influenza viruses and eventually also to novel pandemic influenza viruses has a long history. The ectodomain of the Influenza A Virus M2 Protein has been identified as a possible candidate immunization against influenza. The present study describes the expression of cloned M2 gene in MDCK, HeLa, and COS-7 cells, i.e., in three established eukaryotic cell lines. The expression efficiency was demonstrated by immunofluorescent staining of transfected cells by ELISA, by SDS-PAGE-, and by Western blot-analysis. High level of expression was observed in COS-7 cells. Expression in HeLa and MDCK cells was less efficient. The plasmids constructed in this study may, after modifications, be used for the production of a DNA vaccine. Alternatively the expression product could be refined and used as a purified antigen for the vaccine. Thus, the M2 recombinant protein provides an ideal product for further antigenic, biochemical, structural and functional characterization of the protein and for evaluating its potential for immunodiagnosis and in vaccine studies.
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Células Eucariotas/virología , Expresión Génica , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Animales , Línea Celular , Chlorocebus aethiops , Perros , Vectores Genéticos , Humanos , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Previous studies in non-human primates (NHPs) have shown that beta oscillations (15-30 Hz) of local field potentials (LFPs) in the arm/hand areas of primary motor cortex (MI) propagate as traveling waves across the cortex. These waves exhibited two stereotypical features across animals and tasks: (1) The waves propagated in two dominant modal directions roughly 180° apart, and (2) their propagation speed ranged from 10 to 35 cm/s. It is, however, unknown if such cortical waves occur in the human motor cortex. This study shows that the two properties of propagating beta waves are present in MI of a tetraplegic human patient while he was instructed to perform an instruction delay center-out task using a cursor controlled by the chin. Moreover, we show that beta waves are sustained and have similar properties whether the subject was engaged in the task or at rest. The directions of the successive sustained waves both in the human subject and a NHP subject tended to switch from one dominant mode to the other, and at least in the NHP subject the estimated distance traveled between successive waves traveling into and out of the central sulcus is consistent with the hypothesis of wave reflection between the border of motor and somatosensory cortices. Further, we show that the occurrence of the beta waves is not uniquely tied to periods of increased power in the beta frequency band. These results demonstrate that traveling beta waves in MI are a general phenomenon occurring in human as well as NHPs. Consistent with the NHP data, the dominant directions of the beta LFP waves in human aligned to the proximal to distal gradient of joint representations in MI somatotopy. This consistent finding of wave propagation may imply the existence of a hardwired organization of motor cortex that mediates this spatiotemporal pattern.
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Few studies have investigated how the cortex encodes the preshaping of the hand as an object is grasped, an ethological movement referred to as prehension. We developed an encoding model of hand kinematics to test whether primary motor cortex (MI) neurons encode temporally extensive combinations of joint motions that characterize a prehensile movement. Two female rhesus macaque monkeys were trained to grasp 4 different objects presented by a robot while their arm was held in place by a thermoplastic brace. We used multielectrode arrays to record MI neurons and an infrared camera motion tracking system to record the 3-D positions of 14 markers placed on the monkeys' wrist and digits. A generalized linear model framework was used to predict the firing rate of each neuron in a 4 ms time interval, based on its own spiking history and the spatiotemporal kinematics of the joint angles of the hand. Our results show that the variability of the firing rate of MI neurons is better described by temporally extensive combinations of finger and wrist joint angle kinematics rather than any individual joint motion or any combination of static kinematic parameters at their optimal lag. Moreover, a higher percentage of neurons encoded joint angular velocities than joint angular positions. These results suggest that neurons encode the covarying trajectories of the hand's joints during a prehensile movement.
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Fuerza de la Mano/fisiología , Corteza Motora/fisiología , Movimiento/fisiología , Neuronas/fisiología , Potenciales de Acción/fisiología , Animales , Brazo/fisiología , Fenómenos Biomecánicos , Femenino , Mano/fisiología , Articulaciones/fisiología , Modelos Lineales , Macaca mulattaRESUMEN
Beta oscillations (12-30 Hz) in local field potentials are prevalent in the motor system, yet their functional role within the context of planning a movement is still debated. In this study, a human participant implanted with a multielectrode array in the hand area of primary motor cortex (MI) was instructed to plan a movement using either the second or fourth of five sequentially presented instruction cues. The beta amplitude increased from the start of the trial until the informative (second or fourth) cue, and was diminished afterwards. Moreover, the beta amplitude peaked just prior to each instruction cue and the delta frequency (0.5-1.5 Hz) entrained to the interval between the cues-but only until the informative cue. This result suggests that the beta amplitude and delta phase in MI reflect the subject's engagement with the rhythmically presented cues and work together to enhance sensitivity to predictable and task-relevant visual cues.
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Relojes Biológicos/fisiología , Corteza Motora/fisiología , Neuronas/fisiología , Desempeño Psicomotor/fisiología , Aprendizaje por Asociación/fisiología , Mapeo Encefálico , Señales (Psicología) , Electrodos Implantados , Electrofisiología , Humanos , Intención , Modelos Neurológicos , Movimiento/fisiología , Red Nerviosa/fisiología , Procesamiento de Señales Asistido por Computador , Conducta Espacial/fisiologíaRESUMEN
Primary motor cortex (M1), a key region for voluntary motor control, has been considered a first choice as the source of neural signals to control prosthetic devices for humans with paralysis. Less is known about the potential for other areas of frontal cortex as prosthesis signal sources. The frontal cortex is widely engaged in voluntary behavior. Single-neuron recordings in monkey frontal cortex beyond M1 have readily identified activity related to planning and initiating movement direction, remembering movement instructions over delays, or mixtures of these features. Human functional imaging and lesion studies also support this role. Intraoperative mapping during deep brain stimulator placement in humans provides a unique opportunity to evaluate potential prosthesis control signals derived from nonprimary areas and to expand our understanding of frontal lobe function and its role in movement disorders. This study shows that recordings from small groups of human prefrontal/premotor cortex neurons can provide information about movement planning, production, and decision-making sufficient to decode the planned direction of movement. Thus, additional frontal areas, beyond M1, may be valuable signal sources for human neuromotor prostheses.
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Corteza Motora/patología , Trastornos del Movimiento/patología , Movimiento/fisiología , Neuronas/fisiología , Potenciales de Acción/fisiología , Anciano , Mapeo Encefálico , Femenino , Humanos , Funciones de Verosimilitud , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/fisiopatología , Pruebas Neuropsicológicas , Orientación/fisiología , Valor Predictivo de las Pruebas , Desempeño Psicomotor/fisiologíaRESUMEN
Neuromotor prostheses (NMPs) aim to replace or restore lost motor functions in paralysed humans by routeing movement-related signals from the brain, around damaged parts of the nervous system, to external effectors. To translate preclinical results from intact animals to a clinically useful NMP, movement signals must persist in cortex after spinal cord injury and be engaged by movement intent when sensory inputs and limb movement are long absent. Furthermore, NMPs would require that intention-driven neuronal activity be converted into a control signal that enables useful tasks. Here we show initial results for a tetraplegic human (MN) using a pilot NMP. Neuronal ensemble activity recorded through a 96-microelectrode array implanted in primary motor cortex demonstrated that intended hand motion modulates cortical spiking patterns three years after spinal cord injury. Decoders were created, providing a 'neural cursor' with which MN opened simulated e-mail and operated devices such as a television, even while conversing. Furthermore, MN used neural control to open and close a prosthetic hand, and perform rudimentary actions with a multi-jointed robotic arm. These early results suggest that NMPs based upon intracortical neuronal ensemble spiking activity could provide a valuable new neurotechnology to restore independence for humans with paralysis.