Three-micrometer-diameter needle electrode with an amplifier for extracellular in vivo recordings.

Microscale needle-electrode devices offer neuronal signal recording capability in brain tissue; however, using needles of smaller geometry to minimize tissue damage causes degradation of electrical properties, including high electrical impedance and low signal-to-noise ratio (SNR) recording. We overcome these limitations using a device assembly technique that uses a single needle-topped amplifier package, called STACK, within a device of ∼1 × 1 mm2 Based on silicon (Si) growth technology, a <3-µm-tip-diameter, 400-µm-length needle electrode was fabricated on a Si block as the module. The high electrical impedance characteristics of the needle electrode were improved by stacking it on the other module of the amplifier. The STACK device exhibited a voltage gain of >0.98 (-0.175 dB), enabling recording of the local field potential and action potentials from the mouse brain in vivo with an improved SNR of 6.2. Additionally, the device allowed us to use a Bluetooth module to demonstrate wireless recording of these neuronal signals; the chronic experiment was also conducted using STACK-implanted mice.

Nanoscale-Tipped High-Aspect-Ratio Vertical Microneedle Electrodes for Intracellular Recordings.

Intracellular recording nanoscale electrode devices provide the advantages of a high spatial resolution and high sensitivity. However, the length of nanowire/nanotube-based nanoelectrodes is currently limited to <10 µm long due to fabrication issues for high-aspect-ratio nanoelectrodes. The concept reported here can address the technological limitations by fabricating >100 µm long nanoscale-tipped electrodes, which show intracellular recording capability.

Co-Design Method and Wafer-Level Packaging Technique of Thin-Film Flexible Antenna and Silicon CMOS Rectifier Chips for Wireless-Powered Neural Interface Systems.

In this paper, a co-design method and a wafer-level packaging technique of a flexible antenna and a CMOS rectifier chip for use in a small-sized implantable system on the brain surface are proposed. The proposed co-design method optimizes the system architecture, and can help avoid the use of external matching components, resulting in the realization of a small-size system. In addition, the technique employed to assemble a silicon large-scale integration (LSI) chip on the very thin parylene film (5 µm) enables the integration of the rectifier circuits and the flexible antenna (rectenna). In the demonstration of wireless power transmission (WPT), the fabricated flexible rectenna achieved a maximum efficiency of 0.497% with a distance of 3 cm between antennas. In addition, WPT with radio waves allows a misalignment of 185% against antenna size, implying that the misalignment has a less effect on the WPT characteristics compared with electromagnetic induction.

Closed-loop electrical stimulation to prevent focal epilepsy progression and long-term memory impairment.

Interictal epileptiform discharges (IEDs) are ubiquitously expressed in epileptic networks and disrupt cognitive functions. It is unclear whether addressing IED-induced dysfunction could improve epilepsy outcomes as most therapeutics target seizures. We show in a model of progressive hippocampal epilepsy that IEDs produce pathological oscillatory coupling which is associated with prolonged, hypersynchronous neural spiking in synaptically connected cortex and expands the brain territory capable of generating IEDs. A similar relationship between IED-mediated oscillatory coupling and temporal organization of IEDs across brain regions was identified in human subjects with refractory focal epilepsy. Spatiotemporally targeted closed-loop electrical stimulation triggered on hippocampal IED occurrence eliminated the abnormal cortical activity patterns, preventing spread of the epileptic network and ameliorating long-term spatial memory deficits in rodents. These findings suggest that stimulation-based network interventions that normalize interictal dynamics may be an effective treatment of epilepsy and its comorbidities, with a low barrier to clinical translation. One-Sentence Summary: Targeted closed-loop electrical stimulation prevents spread of the epileptic network and ameliorates long-term spatial memory deficits.

Low-invasive neural recording in mouse models with diabetes via an ultrasmall needle-electrode.

Diabetes is known to cause a variety of complications, having a high correlation with Alzheimer's disease. Electrophysiological recording using a microscale needle electrode is a promising technology for the study, however, diabetic brain tissue is more difficult to record neuronal activities than normal tissue due to these complications including the development of cerebrovascular disease. Here we show an electrophysiological methodology for diabetic db/db mice (+Leprdb/+Leprdb) using a 4-µm-tip diameter needle-electrode device. The needle electrode minimized the tissue injury when compared to a typical larger metal electrode, as confirmed by bleeding during penetration. The proposed electrode device showed both acute and chronic in vivo recording capabilities for diabetic mice while reducing the glial cells' responses. Because of these device characteristics, the 4-µm-tip diameter needle-electrode will allow electrophysiological studies on diabetes models of not only mice, as proven in this study, but also other animals.

Nanoscale tipped microwire arrays enhance electrical trap and depth injection of nanoparticles.

Nanoscale devices have the potential to measure biological tissues as well as individual cells/neurons. However, three-dimensional (3D) multi-site probing remains problematic because only planar-type device designs are applicable to sample surfaces. Herein we report 3D nanoscale electrode tipped microwire arrays with high aspect ratios. A nanoscale tipped wire is formed by isotropic silicon etching to the tip of a vapor-liquid-solid grown silicon microwire. After coating the wire with a metal (e.g., Pt and Au), only the nanotip section can be exposed from the surrounding outer shell (e.g., SiO(2) and parylene) by photoresist spray coating and subsequent cycled photoresist etchings. As a promising device application, we demonstrate the trapping of polystyrene nanoparticles in a solution using a fabricated Au-nanotip wire array. The sharpened nanotip has a 150 nm curvature radius and a 4.2 µm(2) electrode area. The nanotip wires exhibit a locally enhanced trapping performance with a low trapping voltage of 20 mV. Moreover, these trapped nanoparticles can be injected into a soft material (gelatin), demonstrating a multi-site wide-area batch depth injection and an assembly of nanoparticles. Such nanotip wire arrays should be applicable to trap numerous particles, including DNA/molecules attached to Au particles, and may realize injection into biological tissues and individual cells/neurons.

A floating 5 µm-diameter needle electrode on the tissue for damage-reduced chronic neuronal recording in mice.

Microelectrode technology is essential in electrophysiology and has made contributions to neuroscience as well as to medical applications. However, it is necessary to minimize tissue damage associated with needle-like electrode on the brain tissue and the implantation surgery, which makes stable chronic recording impossible. Here, we report on an approach for using a 5 µm-diameter needle electrode, which enables the following of tissue motions, via a surgical method. The electrode is placed on the brain tissue of a mouse with a dissolvable material, reducing the physical stress to the tissue; this is followed by the implantation of the electrode device in the brain without fixing it to the cranium, achieving a floating electrode architecture on the tissue. The electrode shows stable recording with no significant degradation of the signal-to-noise ratios for 6 months, and minimized tissue damage is confirmed compared to that when using a cranium-fixed electrode with the same needle geometry.

Nanoscale-tipped wire array injections transfer DNA directly into brain cells ex vivo and in vivo.

Genetic modification to restore cell functions in the brain can be performed through the delivery of biomolecules in a minimally invasive manner into live neuronal cells within brain tissues. However, conventional nanoscale needles are too short (lengths of ~10 µm) to target neuronal cells in ~1-mm-thick brain tissues because the neuronal cells are located deep within the tissue. Here, we report the use of nanoscale-tipped wire (NTW) arrays with diameters < 100 nm and wire lengths of ~200 µm to address biomolecule delivery issues. The NTW arrays were manufactured by growth of silicon microwire arrays and nanotip formation. This technique uses pinpoint, multiple-cell DNA injections in deep areas of brain tissues, enabling target cells to be marked by fluorescent protein (FP) expression vectors. This technique has potential for use for electrophysiological recordings and biological transfection into neuronal cells. Herein, simply pressing an NTW array delivers and expresses plasmid DNA in multiple-cultured cells and multiple-neuronal cells within a brain slice with reduced cell damage. Additionally, DNA transfection is demonstrated using brain cells ex vivo and in vivo. Moreover, knockdown of a critical clock gene after injecting a short hairpin RNA (shRNA) and a genome-editing vector demonstrates the potential to genetically alter the function of living brain cells, for example, pacemaker cells of the mammalian circadian rhythms. Overall, our NTW array injection technique enables genetic and functional modification of living cells in deep brain tissue areas, both ex vivo and in vivo.

Nanoneedle-Electrode Devices for In Vivo Recording of Extracellular Action Potentials.

Microscale needle-like electrode technologies offer in vivo extracellular recording with a high spatiotemporal resolution. Further miniaturization of needles to nanoscale minimizes tissue injuries; however, a reduced electrode area increases electrical impedance that degrades the quality of neuronal signal recording. We overcome this limitation by fabricating a 300 nm tip diameter and 200 µm long needle electrode where the amplitude gain with a high-impedance electrode (>15 MΩ, 1 kHz) was improved from 0.54 (-5.4 dB) to 0.89 (-1.0 dB) by stacking it on an amplifier module of source follower. The nanoelectrode provided the recording of both local field potential (<300 Hz) and action potential (>500 Hz) in the mouse cortex, in contrast to the electrode without the amplifier. These results suggest that microelectrodes can be further minimized by the proposed amplifier configuration for low-invasive recording and electrophysiological studies in submicron areas in tissues, such as dendrites and axons.

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster.

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.

MUC1 induces metastasis in esophageal squamous cell carcinoma by upregulating matrix metalloproteinase 13.

Esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances because of its high frequency of metastasis. Given the associations of MUC1 with ESCC and tumor metastasis, we explored a potential role of MUC1 in ESCC metastasis. Among 40 ESCC and 20 paired normal tissue specimens examined, we found a significant increase of MUC1 expression in ESCC and more importantly, that expression of MUC1 and MMP13 are strongly correlated in patients who had lymph node metastasis. Studies with cell models indicated that overexpression of MUC1 upregulates the expression of MMP13, leading to increased cell migration. In support of a mode of transcriptional regulation, promoter analysis revealed that MUC1 stimulates MMP13 expression through the Runx-2-binding site. The link of MUC1 to cell motility was further confirmed by the finding that depletion of MUC1 resulted in reduced expression of MMP13 and cell migration, invasion and adhesion. Moreover, the loss of cell metastatic potential was rescued by overexpression of MMP13 completely. Collectively, our findings indicate that MUC1 contributes to ESCC metastasis by stimulating MMP13 expression, suggesting MUC1 as a novel diagnostic biomarker and therapeutic target in ESCC.

Microtube-based electrode arrays for low invasive extracellular recording with a high signal-to-noise ratio.

We report on the development of a microtube electrode array as a neural interface device. To combine the desired properties for the neural interface device, such as low invasiveness with a small needle and a good signal-to-noise ratio in neural recordings, we applied the structure of a glass pipette electrode to each microtube electrode. The device was fabricated as sub-5-microm-diameter out-of-plane silicon dioxide microtube arrays using silicon microneedle templates, which are grown by the selective vapor-liquid-solid method. The microtubes had inner diameters of 1.9-6.4 microm and a length of 25 microm. Impedances ranged from 220 kOmega to 1.55 MOmega, which are less than those for conventional microneedles. In addition, the microtube electrodes had less signal attenuation than conventional microneedle electrodes. We confirmed that the effects of parasitic capacitances between neighboring microtubes and channels were sufficiently small using a test signal. Finally, neural responses evoked from a rat peripheral nerve were recorded in vivo using a microtube electrode to confirm that this type of electrode can be used for both electrophysiological measurements and as a neural interface device.

Nanoscale sharpening tips of vapor-liquid-solid grown silicon microwire arrays.

We developed out-of-plane, high aspect ratio, nanoscale tip silicon microwire arrays for application to penetrating, multisite, nanoscale biological sensors. Silicon microwire arrays selectively grown by gold-catalyzed vapor-liquid-solid growth of silicon can be formed to create sharpened nanotips with a tip diameter of less than 100 nm by utilizing batch-processed silicon chemical etching for only 1-3 min. The tip angles achieved ranged from 11 degrees to 38 degrees. The nanotip silicon microwires can perform gelatin penetration without wire breakdown, indicating their potential penetrating capability for measurements inside biological tissues.

Rapid Photoinduced Single Cell Detachment from Gold Nanoparticle-Embedded Collagen Gels with Low Denaturation Temperature.

Cell Separation is important in various biomedical fields. We have prepared gold nanoparticle (AuNP)-embedded collagen gels as a visible-light-responsive cell scaffold in which photoinduced single cell detachment occurs through local thermal denaturation of the collagen gel via the photothermal effect of AuNP. Physicochemical properties of collagen materials depend on the origin of the collagen and the presence of telopeptides. In this study, we prepared various AuNP-embedded collagen gels by using different collagen materials with and without the telopeptides to compare their thermal denaturation properties and photoinduced single cell detachment behaviors. Cellmatrix type I-C without telopeptides exhibited a lower denaturation temperature than Cellmatrix type I-A and Atelocell IAC, as examined by Fourier transform infrared (FTIR) spectroscopy, rheological analysis, and sol-gel transition observation. Three-dimensional (3D) laser microscopic imaging revealed that collagen fibers shrank in Cellmatrix type I-A upon heating, but collagen fibers disappeared in Cellmatrix type I-C upon heating. Cells cultured on the Cellmatrix type I-C-based AuNP-embedded collagen gel detached with shorter photoirradiation than on the Cellmatrix type I-A-based AuNP-embedded collagen gel, suggesting that collagen gels without telopeptides are suitable for a photoinduced single cell detachment system.

The novel structure of a pyridoxal 5'-phosphate-dependent fold-type I racemase, alpha-amino-epsilon-caprolactam racemase from Achromobacter obae.

Alpha-amino-epsilon-caprolactam (ACL) racemase (ACLR) from Achromobacter obae catalyzes the interconversion of l- and d-ACL. ACLR belongs to the fold-type I group of pyridoxal 5'-phosphate (PLP) dependent enzymes. In this study, the first crystal structures of a fold-type I racemase are solved for the native form and epsilon-caprolactam-complexed form of ACLR at 2.21 and 2.40 A resolution, respectively. Based on the location of epsilon-caprolactam in the complex structure, the substrate-binding site is assigned between Trp49 and Tyr137. The carboxyl group of Asp210 is a reasonable candidate that recognizes the nitrogen atom of a lactam or amide in the substrate. Based on a structural comparison with fold-type III alanine racemase, Tyr137 is potentially the acid/base catalytic residue that is essential for the two-base racemization mechanism. The overall structure of ACLR is similar to that of fold-type I enzymes. A structural comparison with these enzymes explains the different reaction specificities.

Antitumor activity of TMPyP4 interacting G-quadruplex in retinoblastoma cell lines.

To investigate the molecular mechanism of the antitumor activity of the cationic porphyrin 5, 10, 15, 20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4) in retinoblastoma cell lines, Y79 and WERI-Rb1 cells were treated with TMPyP4 for 0-72 h, after which growth inhibition, modulation of the cell cycle and the induction of apoptosis were examined. In addition, the effect of TMPyP4 on the susceptibility to irradiation was evaluated in Y79 and WERI-Rb1 cells. In vitro telomeric repeat amplification protocol assay showed TMPyP4 (10-100 microM) directly blocked telomerase elongation, suggesting that TMPyP4 can form stable guanine (G)-quadruplexes in extending telomere repeats in substrate oligonucleotides. The antiproliferative activities of TMPyP4 assessed with the MTS assay and expressed in terms of IC(50): Y79 cells, 60 microM; WERI-Rb1 cells, 45 microM. Treatment with TMPyP4 at doses of 10, 20, 50 or 100 microM for 48 or 72 h significantly inhibited the growth of Y79 and WERI-Rb1 cells. Apoptosis, as assessed with CaspACE FITC-VAD-FMK, was induced by TMPyP4 in a dose-dependent manner. Induction of apoptosis by TMPyP4 was associated with increased expression of phosphorylated DNA damage response factor H2AX (Ser139), phosphorylated p53 (Ser46) protein and activation of mitogen-activated protein kinases in Y79 and WERI-Rb1 cells. Moreover, TMPyP4 significantly enhanced the susceptibility to irradiation in both cell lines. This study provides insight into the molecular mechanism of the antitumor effects of TMPyP4. G-quadruplex structure may be a potential therapeutic target in retinoblastoma.

Out-of-plane microtube arrays for drug delivery--liquid flow properties and an application to the nerve block test.

We have proposed fabricating very fine out-of-plane silicon-dioxide microtube arrays using a selective vapor-liquid-solid (VLS) growth technique and microfabrication processes. In this study, we elucidated the liquid-flow properties of microtubes with different inner diameters. Our fabricated microtubes were 0.5 microm in wall thickness; 20 microm in height; and either 2.5 microm, 4.1 microm, 4.6 microm, or 6.4 microm in inner diameter. We determined the relationship between the flow pressure and the liquid flow rate through the microtube. We also conducted a nerve block test, in which a microtube with 4.6 microm inner diameter was used to administer lidocaine solution (Na channel blocker) to the rat sciatic nerve. This successful test represents the first reported use of a microtube for drug delivery to the peripheral nerve of a rat. We conclude that the proposed microtube array and its fabrication process might contribute to developing pharmacological devices.

Donut-Shaped Stretchable Kirigami: Enabling Electronics to Integrate with the Deformable Muscle.

Electronic devices used to record biological signals are important in neuroscience, brain-machine interfaces, and medical applications. Placing electronic devices below the skin surface and recording the muscle offers accurate and robust electromyography (EMG) recordings. The device stretchability and flexibility must be similar to the tissues to achieve an intimate integration of the electronic device with the biological tissues. However, conventional elastomer-based EMG electrodes have a Young's modulus that is ≈20 times higher than that of muscle. In addition, these stretchable devices also have an issue of displacement on the tissue surface, thereby causing some challenges during accurate and robust EMG signal recordings. In general, devices with kirigami design solve the issue of the high Young's modulus of conventional EMG devices. In this study, donut-shaped kirigami bioprobes are proposed to reduce the device displacement on the muscle surface. The fabricated devices are tested on an expanding balloon and they show no significant device (microelectrode) displacement. As the package, the fabricated device is embedded in a dissolvable material-based scaffold for easy-to-use stretchable kirigami device in an animal experiment. Finally, the EMG signal recording capability and stability using the fabricated kirigami device is confirmed in in vivo experiments without significant device displacements.

A Magnetically Assembled High-Aspect-Ratio Needle Electrode for Recording Neuronal Activity.

Microelectrode devices, which enable the detection of neuronal signals in brain tissues, have made significant contributions in the field of neuroscience and the brain-machine interfaces. To further develop such microelectrode devices, the following requirements must be met: i) a fine needle's diameter (<30 µm) to reduce damage to tissues; ii) a long needle (e.g., ≈1 mm for rodents and ≈2 mm for macaques); and iii) multiple electrodes to achieve high spatial recording (<100 µm in pitch). In order to meet these requirements, this study herein reports an assembly technique for high-aspect-ratio microneedles, which employs a magnet. The assembly is demonstrated, in which nickel wires of length 750 µm and diameter 25 µm are produced on a silicon substrate. The impedance magnitude of the assembled needle-like electrode measured at 1 kHz is 5.6 kΩ, exhibiting output and input signal amplitudes of 96.7% at 1 kHz. To confirm the recording capability of the fabricated device, neuronal signal recordings are performed using mouse cerebra in vivo. The packaged single microneedle electrode penetrates the barrel field in the primary somatosensory cortex of the mouse and enables the detection of evoked neuronal activity of both local field potentials and action potentials.

MUC1 oncoprotein promotes growth and survival of human multiple myeloma cells.

The MUC1 oncoprotein is aberrantly expressed in human multiple myeloma cells by mechanisms that are not understood. Moreover, the functional role of MUC1 in multiple myeloma is not known. The present studies demonstrate that the MUC1 gene locus is amplified in multiple myeloma cell lines and in primary cells from patients. The KMS28PE multiple myeloma cell line, which was found to have MUC1 gene amplification, was stably silenced for MUC1 using different siRNAs. Silencing MUC1 was associated with a decrease in nuclear beta-catenin levels, consistent with the function of MUC1 in stabilizing beta-catenin. MUC1 is also known to activate the IKKbeta-->NF-kappaB pathway and KMS28PE cells silenced for MUC1 were found to have downregulation of IKKbeta and IkappaBalpha phosphorylation, and decreased nuclear targeting of NF-kappaB p65. The results also demonstrate that MUC1: i) contributes to KMS28PE cell proliferation, and ii) protects against apoptosis and loss of self-renewal in the response to melphalan and dexamethasone. These findings indicate that MUC1 activates the beta-catenin and NF-kappaB pathways in multiple myeloma cells and contributes to their growth and survival.