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Recently, miraculous therapy approaches involving adeno-associated virus (AAV) for incurable diseases such as spinal muscular atrophy and inherited retinal dysfunction have been introduced. Nonreplicative, nonpathogenic, low rates of chromosome insertional properties and the existence of neutralizing antibodies are main safety reasons why the FDA approved its use in gene delivery. To date, AAV production always results in a mixture of nontherapeutic (empty) and therapeutic (DNA-loaded) full capsids (10-98%). Such existence of empty viral particles inevitably increases viral doses to human. Thus, the rapid monitoring of empty capsids and reducing the empty-to-full ratio are critical in AAV science. However, transmission electron microscopy (TEM) is the primary tool for distinguishing between empty and full capsids, which creates a research bottleneck because of instrument accessibility and technical difficulty. Herein, we demonstrate that atomic force microscopy (AFM) can be an alternative tool to TEM. The simple, noncontact-mode imaging of AAV particles allows the distinct height difference between full capsids (â¼22 nm) and empty capsids (â¼16 nm). The sphere-to-ellipsoidal morphological distortion observed for empty AAV particles clearly distinguishes them from full AAV particles. Our study indicates that AFM imaging can be an extremely useful, quality-control tool in AAV particle monitoring, which is beneficial for the future development of AAV-based gene therapy.
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Cápside , Dependovirus , Humanos , Dependovirus/genética , Microscopía de Fuerza Atómica , Vectores Genéticos , ADNRESUMEN
Free space optical (FSO) communication can support various unmanned aerial vehicles' (UAVs) applications that require large capacity data transmission. In order to perform FSO communication between two terminals, it is essential to employ a pointing, acquisition, and tracking (PAT) system with an efficient and optimal performance. We report on the development of a common optical-path-based FSO communication system, tailored for applications in UAVs. The proposed system is equipped with a quadrant photodiode (QPD)-based PAT system without an additional beacon beam subsystem. The presented approach reduced the structural complexity and improved the tracking efficiency for the same size, weight, and power (SWaP). To achieve a robust FSO link in a dynamic UAV environment, the observability and controllability were obtained based on the linearized control according to the incident beam size on the QPD, which was verified by optical simulation and experiments. As a result, the QPD-based PAT system for implementing FSO links demonstrated an up to 4.25 times faster tracking performance. Moreover, the FSO link experimentally confirmed the 1.25 Gbps full-duplex error-free communication at a 50 m distance.
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Gene therapy technologies are inevitably required to boost the therapeutic performance of cell therapies; thus, validating the efficacy of gene carriers specifically used for preparing cellular therapeutics is a prerequisite for evaluating the therapeutic capabilities of gene and cell combinatorial therapies. Herein, the efficacy of a recombinant adeno-associated virus derivative (rAAVr3.45) was examined to evaluate its potential as a gene carrier for genetically manipulating interleukin-10 (IL10)-secreting human neural stem cells (hNSCs) that can potentially treat ischemic injuries or neurological disorders. Safety issues that could arise during the virus preparation or viral infection were investigated; no replication-competent AAVs were detected in the final cell suspensions, transgene expression was mostly transient, and no severe interference on endogenous gene expression by viral infection occurred. IL10 secretion from hNSCs infected by rAAVr3.45 encoding IL10 did not alter the transcriptional profile of any gene by more than threefold, but the exogenously boosted IL10 was sufficient to provoke immunomodulatory effects in an ischemic brain injury animal model, thereby accelerating the recovery of neurological deficits and the reduction of brain infarction volume. This study presents evidence that rAAVr3.45 can be potentially used as a gene carrier to prepare stem cell therapeutics.
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Isquemia Encefálica/terapia , Dependovirus/genética , Terapia Genética/métodos , Interleucina-10/genética , Células-Madre Neurales/trasplante , Trasplante de Células Madre/métodos , Animales , Células Cultivadas , Terapia Genética/efectos adversos , Células HEK293 , Humanos , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos ICR , Células-Madre Neurales/metabolismo , Trasplante de Células Madre/efectos adversosRESUMEN
Metasurfaces are two dimensional arrays of artificial subwavelength resonators, which can manipulate the amplitude and phase profile of incident electromagnetic fields. To date, limited progress has been achieved in realizing reconfigurable phase control of incident waves using metasurfaces. Here, an active metasurface is presented, whose resonance frequency can be tuned by employing insulator to metal transition in vanadium dioxide. By virtue of the phase jump accompanied by the resonance frequency tuning, the proposed metasurface acts as a phase shifter at THz frequency. It is further demonstrated that by appropriately tailoring the anisotropy of the metasurface, the observed phase shift can be used to switch the transmitted polarization from circular to approximately linear. This work thus shows potential for reconfigurable phase and polarization control at THz frequencies using vanadium dioxide based frequency tunable metasurfaces.
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Tissue adhesives, which inherently serve as wound sealants or as hemostatic agents, can be further augmented to acquire crucial functions as scaffolds, thereby accelerating wound healing or elevating the efficacy of tissue regeneration. Herein, multifunctional adherent fibrous matrices, acting as self-adhesive scaffolds capable of cell/gene delivery, were devised by coaxially electrospinning poly(caprolactone) (PCL) and poly(vinylpyrrolidone) (PVP). Wrapping the building block PCL fibers with the adherent PVP layers formed film-like fibrous matrices that could rapidly adhere to wet biological surfaces, referred to as fibrous layered matrix (FiLM) adhesives. The inclusion of ionic salts (i.e., dopamine hydrochloride) in the sheath layers generated spontaneously multilayered fibrous adhesives, whose partial layers could be manually peeled off, termed derivative FiLM (d-FiLM). In the context of scaffolds/tissue adhesives, both FiLM and d-FiLM demonstrated almost identical characteristics (i.e., sticky, mechanical, and performances as cell/gene carriers). Importantly, the single FiLM-process can yield multiple sets of d-FiLM by investing the same processing time, materials, and labor required to form a single conventional adhesive fibrous mat, thereby highlighting the economic aspects of the process. The FiLM/d-FiLM offer highly impacting contributions to many biomedical applications, especially in fields that require urgent aids (e.g., endoscopic surgeries, implantation in wet environments, severe wounds).
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Poliésteres/química , Adhesivos Tisulares/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Cicatrización de Heridas , Animales , Materiales Biocompatibles/química , Supervivencia Celular/efectos de los fármacos , Dependovirus/genética , Sistemas de Liberación de Medicamentos , Femenino , Vectores Genéticos/administración & dosificación , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Células Jurkat , Ensayo de Materiales , Ratones , Ratones Endogámicos ICR , Células 3T3 NIH , PorosidadRESUMEN
Supercapacitors, based on fast ion transportation, are specialized to provide high power, long stability, and efficient energy storage using highly porous electrode materials. However, their low energy density excludes them from many potential applications that require both high energy density and high power density performances. Using a scalable nanoporous graphene synthesis method involving an annealing process in hydrogen, here we show supercapacitors with highly porous graphene electrodes capable of achieving not only a high power density of 41 kW kg-1 and a Coulombic efficiency of 97.5%, but also a high energy density of 148.75 Wh kg-1. A high specific gravimetric and volumetric capacitance (306.03 F g-1 and 64.27 F cm-3) are demonstrated. The devices can retain almost 100% capacitance after 7000 charging/discharging cycles at a current density of 8 A g-1. The superior performance of supercapacitors is attributed to their ideal pore size, pore uniformity, and good ion accessibility of the synthesized graphene.
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Advanced technologies that can mimic hierarchical architectures found in nature can provide pivotal clues for elucidating numerous biological mechanisms. Herein, a novel technology, spontaneous organization of numerous-layer generation by electrospray (SpONGE), was developed to create self-assembled and multilayered fibrous structures. The simple inclusion of salts in a polymer solution prior to electrospraying was key to mediating the structural versatilities of the fibrous structures. The SpONGE matrix demonstrated great potential as a crucial building block capable of inducing sequential, localized drug delivery or orchestrating cellular distribution inâ vivo, thereby expanding its scope of use to cover a variety of biomedical applications.
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Low insertion-loss single-layer and bilayer metamaterial filters in terahertz (THz) frequency regime were demonstrated on top of low cost flexible Scotch tape by utilizing pattern transfer method. The transmittance of the flexible 51-µm-thick Scotch tape was found out to be higher than 0.85 in the range of 0.2 to 3 THz, which is excellent for the substrate materials for THz applications. Free standing filters exhibited record low insertion loss of 0.6 dB and band rejection ratio as high as 30 dB. The resonance reflection characteristics of the bilayer filters were maintained when they were attached on top of curved PET bottle or metallic surfaces, providing promising application in THz identifications.
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Electrospun fibrous mats have emerged as powerful tissue engineering scaffolds capable of providing highly effective and versatile physical guidance, mimicking the extracellular environment. However, electrospinning typically produces a sheet-like structure, which is a major limitation associated with current electrospinning technologies. To address this challenge, highly porous, volumetric hydrogel-hybrid fibrous scaffolds were fabricated by one Taylor cone-based side-by-side dual electrospinning of poly (ε-caprolactone) (PCL) and poly (vinyl pyrrolidone) (PVP), which possess distinct properties (i.e., hydrophobic and hydrogel properties, respectively). Immersion of the resulting scaffolds in water induced spatial tortuosity of the hydrogel PVP fibers while maintaining their aligned fibrous structures in parallel with the PCL fibers. The resulting conformational changes in the entire bicomponent fibers upon immersion in water led to volumetric expansion of the fibrous scaffolds. The spatial fiber tortuosity significantly increased the pore volumes of electrospun fibrous mats and dramatically promoted cellular infiltration into the scaffold interior both in vitro and in vivo. Harmonizing the flexible PCL fibers with the soft PVP-hydrogel layers produced highly ductile fibrous structures that could mechanically resist cellular contractile forces upon in vivo implantation. This facile dual electrospinning followed by the spatial fiber tortuosity for fabricating three-dimensional hydrogel-hybrid fibrous scaffolds will extend the use of electrospun fibers toward various tissue engineering applications.
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Materiales Biocompatibles/química , Hidrogeles/química , Nanofibras/química , Poliésteres/química , Povidona/química , Andamios del Tejido/química , Animales , Ratones , Células 3T3 NIH , Porosidad , Ingeniería de TejidosRESUMEN
We have investigated THz radiation characteristics along different directions, either reflective or along lateral by using InAs-based heterostructures. Firstly, we demonstrate the phase shift with InAs layer thickness, revealing the change of dominant THz wave generation mechanism along both directions. Along the lateral direction, the time-domain signals in thin InAs epilayers showed an abrupt phase and amplitude change at certain time delays which suggest the interference between two rays at the photoconductive switch. This behavior was further substantiated by the multiple cavity modes in Fourier-transformed spectra and by the amplitude variation with excitation spot displacement.
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The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.
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Cápside , Parvovirinae , Humanos , Células HEK293 , Vectores Genéticos/genética , Dependovirus/genética , Plásmidos/genética , Proteínas de la Cápside/genética , Parvovirinae/genéticaRESUMEN
BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIBI) is a significant contributor to neonatal mortality and long-term neurodevelopmental disability, characterized by massive neuronal loss and reactive astrogliosis. Current therapeutic approaches for neonatal HIBI have been limited to general supportive therapy because of the lack of methods to compensate for irreversible neuronal loss. This study aimed to establish a feasible regenerative therapy for neonatal HIBI utilizing in vivo direct neuronal reprogramming technology. METHODS: Neonatal HIBI was induced in ICR mice at postnatal day 7 by permanent right common carotid artery occlusion and exposure to hypoxia with 8% oxygen and 92% nitrogen for 90 min. Three days after the injury, NeuroD1 was delivered to reactive astrocytes of the injury site using the astrocyte-tropic adeno-associated viral (AAV) vector AAVShH19. AAVShH19 was engineered with the Cre-FLEX system for long-term tracking of infected cells. RESULTS: AAVShH19-mediated ectopic NeuroD1 expression effectively converted astrocytes into GABAergic neurons, and the converted cells exhibited electrophysiological properties and synaptic transmitters. Additionally, we found that NeuroD1-mediated in vivo direct neuronal reprogramming protected injured host neurons and altered the host environment, i.e., decreased the numbers of activated microglia, reactive astrocytes, and toxic A1-type astrocytes, and decreased the expression of pro-inflammatory factors. Furthermore, NeuroD1-treated mice exhibited significantly improved motor functions. CONCLUSIONS: This study demonstrates that NeuroD1-mediated in vivo direct neuronal reprogramming technology through AAV gene delivery can be a novel regenerative therapy for neonatal HIBI.
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A high aspect ratio conical sub-wavelength structure (SWS) was designed by using rigorous coupled-wave analysis (RCWA) method and was realized on polymethyl methacrylate (PMMA) film using a stamping technique. The silicon template containing a hexagonal array of conical holes with a period of 350 nm and an aspect ratio of 2.8 was fabricated by electron-beam (e-beam) lithography followed by a two-step etching process. The SWS with a high aspect ratio was easily transferred from the fabricated silicon template to PMMA film using the stamping method. The replicated PMMA SWS has an array of cones with nanoscale tips and an aspect ratio higher than 2.8. The average reflectance and transmittance of the PMMA film with the conical SWS in the wavelength ranging from 500 and 1500 nm was improved from 7.1 and 91.1% to 4.3 and 94.2%, respectively, as compared to flat PMMA film.
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Impresión Molecular/métodos , Nanopartículas/química , Nanopartículas/ultraestructura , Polimetil Metacrilato/química , Refractometría/instrumentación , Resonancia por Plasmón de Superficie/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
This study describes a simple, versatile approach for developing a nonviral gene carrier by adopting the highly efficient gene delivery properties of the adeno-associated virus (AAV). Specific viral peptides (r3.45_hepBD) extracted from AAV r3.45, which directly evolved to improve gene delivery capabilities in many cell types, were conjugated onto branched polyethylenimine (PEI) to form hybrid gene carriers. AAV r3.45 carries a sequence insertion (LATQVGQKTA; r3.45) within the heparin-binding domain (LQRGNRQA; hepBD), which ultimately comprises a novel sequence (LQRGNLATQVGQKTARQA; r3.45_hepBD) on the capsid. This sequence is hypothesized to be a crucial cue to enhance gene delivery efficiency. Consequently, the intimate interactions of the conjugated r3.45_hepBD with the glycosaminoglycans, including chondroitin sulfate, resulted in significantly enhanced cellular transfection of DNA/PEI-r3.45_hepBD complexes. The successful establishment of a nonviral system that is built with novel peptides will provide a powerful means for developing a substantial number of gene therapy applications.
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Dependovirus , Vectores Genéticos/genética , Transfección/métodos , Proteínas Virales/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Supervivencia Celular , Endocitosis , Terapia Genética/métodos , Células HEK293 , Células HeLa , Humanos , Ratones , Transducción Genética , Proteínas Virales/metabolismoRESUMEN
Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.
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Dependovirus/genética , Evolución Molecular Dirigida , Marcación de Gen , Vectores Genéticos/genética , Células Madre Pluripotentes/metabolismo , Transducción Genética , Secuencia de Bases , Proteínas de la Cápside/genética , Línea Celular , Roturas del ADN de Doble Cadena , Dependovirus/fisiología , Endonucleasas/genética , Expresión Génica , Biblioteca de Genes , Orden Génico , Técnicas de Transferencia de Gen , Humanos , Datos de Secuencia Molecular , Mutación , Tropismo Viral , Dedos de Zinc/genéticaRESUMEN
A nanometer-scale grating structure with a 60-nm-wide gap and 200-nm-wide ridge has been successfully demonstrated on a silicon-on-insulator substrate by using a 220-nm-thick hydrogen silsesquioxane (HSQ) negative tone electron beam resist. A post exposure baking (PEB) process and hot development process with low concentration (3.5 wt%) of tetramethylammonium hydroxide (TMAH) solution were introduced to realize the grating pattern. To study the effects of post exposure baking on the HSQ resist, Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analyses were carried out. From the FT-IR and XPS analyses, it was verified that a thin SiO2 with high cross-linked network structure was formed on the HSQ surface during the PEB step. This SiO2 layer prevents the formation of unwanted bonds on the HSQ surface, which results in clearly defined grating structures with a 60-nm-gap and 200-nm-wide-ridge on the 220-nm-thick HSQ resist. The nanometer-scale grating pattern was successfully transfered to the 280-nm-thick silicon layer of a silicon-on-insulator (SOI) substrate by using inductively-coupled-plasma-reactive-ion-etching (ICP-RIE).
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The effects of electrode materials (top and bottom) and the operating ambiances (open-air and vacuum) on the MgFx-based resistive random-access memory (RRAM) devices are studied. Experiment results show that the device's performance and stability depend on the difference between the top and bottom electrodes' work functions. Devices are robust in both environments if the work function difference between the bottom and top electrodes is greater than or equal to 0.70 eV. The operating environment-independent device performance depends on the surface roughness of the bottom electrode materials. Reducing the bottom electrodes' surface roughness will reduce moisture absorption, minimizing the impact of the operating environment. Ti/MgFx/p+-Si memory devices with the minimum surface roughness of the p+-Si bottom electrode show operating environment-independent electroforming-free stable resistive switching properties. The stable memory devices show promising data retentions of >104 s in both environments with DC endurance properties of more than 100 cycles.
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A broadband terahertz (THz) absorber consisting of multilayer glass spheres and polydimethylsiloxane (PDMS) was realized. The multilayer glass spheres were deposited by repeating a self-assembly method used to form monolayer glass spheres and by the spin-coating of PDMS to fill the gaps between the glass spheres. The average reflection at the surface of the absorber was 0.8% and the absorbance was higher than 98% in the frequency range between 0.7 to 2.0 THz.
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A hemispherical surface structure was fabricated on a sapphire substrate by utilizing a self-assembly and spin-coating process for a terahertz (THz) antireflection coating. The self-assembled glass spheres and spin-coated material led to a gradual change in the effective refractive index. The aspect ratio of the hemispherical surface structure was controlled easily by adjusting the thickness of the B-staged bisbenzocyclobutene used as a coating. The reflectance of the fabricated hemispherical surface structure, having a period of 140 µm, exhibited low reflectance and low Fabry-Perot resonance in a THz spectral range from 0.1 to 1.9 THz.
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Fenómenos Ópticos , Vidrio/química , Compuestos Policíclicos/química , Propiedades de SuperficieRESUMEN
Integrating viral gene delivery with engineered biomaterials is a promising strategy to overcome a number of challenges associated with virus-mediated gene delivery, including inefficient delivery to specific cell types, limited tropism, spread of vectors to distant sites, and immune responses. Viral vectors can be combined with biomaterials either through encapsulation within the material or immobilization onto a material surface. Subsequent biomaterial-based delivery can increase the vector's residence time within the target site, thereby potentially providing localized delivery, enhancing transduction, and extending the duration of gene expression. Alternatively, physical or chemical modification of viral vectors with biomaterials can be employed to modulate the tropism of viruses or reduce inflammatory and immune responses, both of which may benefit transduction. This review describes strategies to promote viral gene delivery technologies using biomaterials, potentially providing opportunities for numerous applications of gene therapy to inherited or acquired disorders, infectious disease, and regenerative medicine.