Adhesion States Greatly Affect Cellular Susceptibility to Graphene Oxide: Therapeutic Implications for Cancer Metastasis.

Graphene oxide (GO) has received increasing attention in the life sciences because of its potential for various applications. Although GO is generally considered biocompatible, it can negatively impact cell physiology under some circumstances. Here, we demonstrate that the cytotoxicity of GO greatly varies depending on the cell adhesion states. Human HCT-116 cells in a non-adhered state were more susceptible to GO than those in an adherent state. Apoptosis was partially induced by GO in both adhered and non-adhered cells to a similar extent, suggesting that apoptosis induction does not account for the selective effects of GO on non-adhered cells. GO treatment rapidly decreased intracellular ATP levels in non-adhered cells but not in adhered ones, suggesting ATP depletion as the primary cause of GO-induced cell death. Concurrently, autophagy induction, a cellular response for energy homeostasis, was more evident in non-adhered cells than in adhered cells. Collectively, our observations provide novel insights into GO's action with regard to cell adhesion states. Because the elimination of non-adhered cells is important in preventing cancer metastasis, the selective detrimental effects of GO on non-adhered cells suggest its therapeutic potential for use in cancer metastasis.

Effect of Nanosecond Pulsed Currents on Directions of Cell Elongation and Migration through Time-Lapse Analysis.

It is generally known that cells elongate perpendicularly to an electric field and move in the direction of the field when an electric field is applied. We have shown that irradiation of plasma-simulated nanosecond pulsed currents elongates cells, but the direction of cell elongation and migration has not been elucidated. In this study, a new time-lapse observation device that can apply nanosecond pulsed currents to cells was constructed, and software to analyze cell migration was created to develop a device that can sequentially observe cell behavior. The results showed nanosecond pulsed currents elongate cells but do not affect the direction of elongation and migration. It was also found the behavior of cells changes depending on the conditions of the current application.

Disease-associated H58Y mutation affects the nuclear dynamics of human DNA topoisomerase IIß.

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics.

Ankle foot orthosis that prevents slippage for tibial rotation in knee osteoarthritis patients.

Knee osteoarthritis (OA) is a disease caused by age-related muscle weakness, obesity, or sports injury that leads to gait disability due to pain during walking. Knee OA is characterized by abnormal knee joint alignment and rotational dyskinesia, which are believed to worsen the symptoms. We previously developed an ankle orthosis that mechanically induces the rotation of the lower limb in conjunction with that of the ankle joint. This orthosis can effectively correct the alignment of the knee joint. However, slippage between the orthosis and leg can occur during walking, decreasing the corrective force. In this study, we clarify the effect of slippage between the orthosis and body on the correction force of the orthosis, and develop a lower leg tracking mechanism to suppress slippage and minimize reduction of force. The effectiveness of the proposed mechanism was evaluated by three-dimensional motion analysis of gait. Analysis results confirmed that the proposed mechanism was effective in suppressing slippage and improving correction force, demonstrating the effectiveness of the mechanism for knee OA.

System for Operating Electric Wheelchairs Using Only the Remaining Functions of the Thumbs of Muscular Dystrophy Patients.

For patients with muscular dystrophy, a motor dysfunction, who have difficulty operating an electric wheelchair with joysticks, a simplified one-input device is used. However, avoiding obstacles can be time-consuming. In this study, we analyzed the motor functions of the thumb of a patient with severe muscular dystrophy and identified the operations that did not cause physical fatigue. Then, we developed an operation support system to continuously operate. Finally, we conducted experiments comparing the proposed system with the conventional system and verified the effectiveness of the proposed system based on the steering accuracy of the electric wheelchair and the task completion time.

Nucleolar translocation of human DNA topoisomerase II by ATP depletion and its disruption by the RNA polymerase I inhibitor BMH-21.

DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.

A fluorescence method to visualize the nuclear boundary by the lipophilic dye DiI.

Here, we describe a procedure to fluorescently contrast the nuclear boundary using the lipophilic carbocyanine dye DiI in cultured human cells. Our procedure is simple and is applicable to detect nuclear boundary defects, which may be relevant to studies on nuclear envelope dynamics, micronuclei formation and cancer biology. ABBREVIATIONS: DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate; NE: nuclear envelope; RanBP2: Ran-binding protein 2/Nucleoporin 358.

Nanosecond pulsed current under plasma-producing conditions induces morphological alterations and stress fiber formation in human fibrosarcoma HT-1080 cells.

Cold atmospheric plasma (CAP) is a promising means for various biomedical applications, including cancer therapy. Although the biological action of CAP is considered to be brought about by synergistic effects of reactive species and electrical factors of CAP, limited information is currently available on the contribution of electrical factors to CAP-induced cell responses. We have previously demonstrated that nanosecond pulsed current (nsPC) under CAP-producing conditions significantly promoted the motility of human HT-1080 cells. In this study, we explored the effects of nsPC on cell morphology associated with cell motility. We observed that nsPC stimulation caused extended cell shape, membrane protrusion formation, and increased cell surface area, but not cell death induction. nsPC stimulation also caused elevated intracellular ROS and Ca2+. HT-1080 cells can undergo two modes of cell motility, namely mesenchymal and ameboid motility, and we found that morphological features of mesenchymal motility was partly shared with nsPC-stimulated cells. Furthermore, nsPC-stimulated cells had extended stress fibers composed of filamentous actin. Taken together, this study provides a novel insight into the electrical aspect of CAP action, and we speculate that nsPC activates a certain mechanism involving intracellular signaling for stress fiber formation, leading to altered cell morphology and increased cell motility.

Mechanical Orthosis with Rotation Induction to Lower Leg for Patients with Knee Osteoarthritis.

Knee osteoarthritis (Knee-OA) is a disease caused by age-related muscle weakness, obesity, or sports injury, and it has been estimated to occur in approximately half of all people by the age of 85. One of the characteristics of knee-OA is rotation dyskinesia of the knee joint due to the degeneration of the system around the knee. This rotation movement, a key element of walking, is crucial for impact absorption, balanced walking, and stabilization of the knee joint. In the present study, we focused on the rotation of the lower leg relative to the movement of the ankle joint during the walking stance phase, and we developed a mechanical orthosis that induces rotation of the lower leg in conjunction with the movement of the ankle joint mechanically. The mechanical induction of rotation movement uses the movement difference due to the angle change of the inside and outside bars in conjunction with the ankle angle. We verified the effectiveness of the developed orthosis by measuring the amount of rotation and by administering the Womac test in 5 subjects with knee osteoarthritis. The results confirmed the effectiveness of our orthosis.

Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells.

Nanosecond pulsed electric fields (nsPEFs) have gained attention as a novel physical stimulus for life sciences. Although cancer therapy is currently their promising application, nsPEFs have further potential owing to their ability to elicit various cellular responses. This study aimed to explore stimulatory actions of nsPEFs, and we used HL-60 cells that were differentiated into neutrophils under cultured conditions. Exposure of neutrophil-differentiated HL-60 cells to nsPEFs led to the extracellular release of chromosomal DNA, which appears to be equivalent to neutrophil extracellular traps (NETs) that serve as a host defense mechanism against pathogens. Fluorometric measurement of extracellular DNA showed that DNA extrusion was rapidly induced after nsPEF exposure and increased over time. Western blot analysis demonstrated that nsPEFs induced histone citrullination that is the hydrolytic conversion of arginine to citrulline on histones and facilitates chromatin decondensation. DNA extrusion and histone citrullination by nsPEFs were cell type-specific and Ca2+-dependent events. Taken together, these observations suggest that nsPEFs drive the mechanism for neutrophil-specific immune response without infection, highlighting a novel aspect of nsPEFs as a physical stimulus.

Joystick Grip for Electric Wheelchair for Tension-Athetosis-Type Cerebral Palsy.

In Japan, the number of people who have difficulty walking has been increasing with the rise in the aging population and that of people with physical disabilities. Individuals with athetosis-type cerebral palsy may use electric wheelchairs due to abnormal walking. However, since they have problems with fine motor control, including the occurrence of involuntary movements and difficulty maintaining posture, they have difficulty intentionally controlling their hand movements. Therefore, they cannot operate a joystick, even if they desire to use electric wheelchairs, and there are risks of accidents. In this study, by considering the arch structure of hand, we developed a new joystick grip that enables the suppression of involuntary movement. We evaluated our proposed grip by comparing running stability with a conventional grip, and demonstrated the effectiveness of proposed method.

Robotic prosthesis that maintains flexion posture.

The number of lower limb amputations in Japan has recently been increasing. Lower limb amputation is classified based on the amputated part,and its two main types are below-knee (28.4%) and above-knee (58.8%). Especially for patients with above-knee amputation, an above-knee prosthesis with artificial knee and ankle joints is commonly applied. This prostheses also vary according to an individual's activity level. Thus there are varieties of uses of above-knee prostheses. Therefore, various knee joints are being developed corresponding to various activity levels. Passive multi linkage-type knee joints aim to guarantee the stability of the stance phase during walking, and electronically controlled knee joints use a hydraulic cylinder to provide stability in both the swing and stance phases as well as during slope walking and so on. As described above, many knee joints focused on walking are commercially available. But in recent years knee joints focusing on other everyday activities, such as sitting or rising, are being developed. However, there has been no assistive prosthesis designed to enable the standing action involved in stopping in a state where the knee remains slightly flexed. In this study, we developed a robotic prosthesis to provide stability during standing as well as during normal walking. A ratchet mechanism was used for the knee joint, and it reproduced the flexion extension motion of the knee. We also tried to electronically control the movement of the claw of the ratchet mechanism based on the state transition model by finite state machine. Furthermore, the effectiveness of the developed robot prosthesis was shown in a walking experiment by a healthy person.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells.

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

Dynamic behavior of DNA topoisomerase IIß in response to DNA double-strand breaks.

DNA topoisomerase II (Topo II) is crucial for resolving topological problems of DNA and plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation. Although DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated. Here, we show the dynamic behavior of human Topo IIß in response to DNA double-strand breaks (DSBs), which is the most harmful form of DNA damage. Live cell imaging coupled with site-directed DSB induction by laser microirradiation demonstrated rapid recruitment of EGFP-tagged Topo IIß to the DSB site. Detergent extraction followed by immunofluorescence showed the tight association of endogenous Topo IIß with DSB sites. Photobleaching analysis revealed that Topo IIß is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIß mobility and thereby prevented Topo IIß recruitment to DSBs. Furthermore, Topo IIß knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo IIß in HR-mediated DSB repair. Taken together, these results highlight a novel aspect of Topo IIß functions in the cellular response to DSBs.

An assistance device to help people with trunk impairment maintain posture.

People with trunk impairment cannot lean forward because of the dysfunction of the trunk resulting from events such as cervical cord injury (CCI). It is therefore difficult for such people to work at a table because they may easily fall from their wheelchair, and it is also hard for them to return to their original position. This limits the activities of daily living (ADLs) of people with trunk impairment. These problems can be solved to some extent with equipment such as a wheelchair belt or a spinal orthosis that can help the person to maintain his or her posture. However, people cannot move freely with this equipment. Furthermore, if this equipment is used for a long time, there is a risk of physical pain and skin issues. In this study, we developed a device that assists the trunk of people with trunk impairment when they lean forward. This device supports people with trunk impairment so that they may take their meals at the table and prevents them from falling over their wheelchair without hindering their daily performance when they are sitting normally. The effectiveness of our proposed device was verified by experiments involving having a meal, operating a wheelchair, and colliding with a curb. Our device can help people with trunk impairment by improving their ADLs and quality of life (QOL).

Calcium-dependent activation of transglutaminase 2 by nanosecond pulsed electric fields.

Exposure of cultured human cells to nanosecond pulsed electric fields (nsPEFs) elicits various cellular events, including Ca2+ influx and cell death. Recently, nsPEFs have been regarded as a novel physical treatment useful for biology and medicine, but the underlying mechanism of action remains to be fully elucidated. In this study, we investigated the effect of nsPEFs on transglutaminases (TGs), enzymes that catalyze covalent protein modifications such as protein-protein crosslinking. Cellular TG activity was monitored by conjugation of cellular proteins with biotin-cadaverine, a cell-permeable pseudosubstrate for TGs. We applied nsPEFs to HeLa S3 cells and found that overall catalytic activity of cellular TGs was greatly increased in a Ca2+-dependent manner. The Ca2+ ionophore ionomycin significantly augmented nsPEF-induced TG activation, further supporting the importance of Ca2+. Among human TG family members, TG2 is known to be the most ubiquitously expressed, and its catalytic activity requires elevated intracellular Ca2+. Given the requirement of Ca2+ for TG activation by nsPEFs, we performed depletion of TG2 by RNA interference (RNAi). We observed that TG2 RNAi suppressed the nsPEF-induced TG activation and partially alleviated the cytotoxic effects of nsPEFs. These findings demonstrate that TG2 activation is a Ca2+-dependent event in nsPEF-exposed cells and exerts negative effects on cell physiology.

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin-induced neutrophil extracellular trap-osis (NETosis)-like cell death in cultured human leukemia cells.

We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR.

Different involvement of extracellular calcium in two modes of cell death induced by nanosecond pulsed electric fields.

Exposure of cultured cells to nanosecond pulsed electric fields (nsPEFs) induces various cellular responses, including the influx of extracellular Ca2+ and cell death. Recently, nsPEFs have been regarded as a novel means of cancer therapy, but their molecular mechanism of action remains to be fully elucidated. Here, we demonstrate the involvement of extracellular Ca2+ in nsPEF-induced cell death. Extracellular Ca2+ was essential for necrosis and consequent poly(ADP-ribose) (PAR) formation in HeLa S3 cells. Treatment with a Ca2+ ionophore enhanced necrosis as well as PAR formation in nsPEF-exposed HeLa S3 cells. In the absence of extracellular Ca2+, HeLa S3 cells were less susceptible to nsPEFs and exhibited apoptotic proteolysis of caspase 3 and PARP-1. HeLa S3 cells retained the ability to undergo apoptosis even after nsPEF exposure but instead underwent necrosis, suggesting that necrosis is the preferential mode of cell death. In K562 and HEK293 cells, exposure to nsPEFs resulted in the formation of necrosis-associated PAR, whereas Jurkat cells exclusively underwent apoptosis independently of extracellular Ca2+. These observations demonstrate that the mode of cell death induced by nsPEFs is cell-type dependent and that extracellular Ca2+ is a critical factor for nsPEF-induced necrosis.

A wheelchair operation assistance control for a wearable robot using the user's residual function.

Cervical Cord Injury (CCI) is a dysfunction of the upper limb. In an individual with C5-level CCI, which is the most frequent of all eight levels, force can be applied in the direction of flexion by the biceps brachii, but extension force cannot be applied because of the triceps brachii paralysis. Persons with C5-level CCI therefore cannot operate a wheelchair up an incline and over carpet. In this study, we estimated the wheelchair velocity during elbow flexion depending on the angular velocity of the elbow. A wearable assistive robot can assist with the elbow extension movement using this estimated velocity while the wheelchair is being operated.