Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway.

The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK) via the MEK-ERK pathway in ligand- and tyrosine kinase-independent manners. Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.

Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy.

Physical inactivity gives rise to numerous diseases and organismal dysfunctions, particularly those related to aging. Musculoskeletal disorders including muscle atrophy, which can result from a sedentary lifestyle, aggravate locomotive malfunction and evoke a vicious circle leading to severe functional disruptions of vital organs such as the brain and cardiovascular system. Although the significance of physical activity is evident, molecular mechanisms behind its beneficial effects are poorly understood. Here, we show that massage-like mechanical interventions modulate immobilization-induced pro-inflammatory responses of macrophages in situ and alleviate muscle atrophy. Local cyclical compression (LCC) on mouse calves, which generates intramuscular pressure waves with amplitude of 50 mmHg, partially restores the myofiber thickness and contracting forces of calf muscles that are decreased by hindlimb immobilization. LCC tempers the increase in the number of cells expressing pro-inflammatory proteins, tumor necrosis factor-α and monocyte chemoattractant protein-1 (MCP-1), including macrophages in situ The reversing effect of LCC on immobilization-induced thinning of myofibers is almost completely nullified when macrophages recruited from circulating blood are depleted by administration of clodronate liposomes. Furthermore, application of pulsatile fluid shear stress, but not hydrostatic pressure, reduces the expression of MCP-1 in macrophages in vitro Together with the LCC-induced movement of intramuscular interstitial fluid detected by µCT analysis, these results suggest that mechanical modulation of macrophage function is involved in physical inactivity-induced muscle atrophy and inflammation. Our findings uncover the implication of mechanosensory function of macrophages in disuse muscle atrophy, thereby opening a new path to develop a novel therapeutic strategy utilizing mechanical interventions.

Interstitial-fluid shear stresses induced by vertically oscillating head motion lower blood pressure in hypertensive rats and humans.

The mechanisms by which physical exercise benefits brain functions are not fully understood. Here, we show that vertically oscillating head motions mimicking mechanical accelerations experienced during fast walking, light jogging or treadmill running at a moderate velocity reduce the blood pressure of rats and human adults with hypertension. In hypertensive rats, shear stresses of less than 1 Pa resulting from interstitial-fluid flow induced by such passive head motions reduced the expression of the angiotensin II type-1 receptor in astrocytes in the rostral ventrolateral medulla, and the resulting antihypertensive effects were abrogated by hydrogel introduction that inhibited interstitial-fluid movement in the medulla. Our findings suggest that oscillatory mechanical interventions could be used to elicit antihypertensive effects.

Morphology Control via RAFT Emulsion Polymerization-Induced Self-Assembly: Systematic Investigation of Core-Forming Blocks.

Polymerization-induced self-assembly (PISA) is a useful formulation for readily obtaining nanoparticles from block copolymers in situ. Reversible addition-fragmentation chain-transfer (RAFT) emulsion polymerization is utilized as one of the PISA formulations. Various factors have so far been investigated for obtaining nonspherical particles via RAFT emulsion polymerization, such as the steric structure of the shell, the glass-transition temperature (T g) of the core-forming block, and the water solubility of the core-forming monomer. This study focuses on core-forming blocks without changing the structure of the shell-forming block. In particular, we elucidate the balance between T g for the core-forming block and the water solubility of the core monomer. A series of alkyl methacrylates, such as methyl methacrylate (MMA), ethyl methacrylate (EMA), and n-propyl methacrylate (PrMA), are emulsion-polymerized in the presence of a poly[poly(ethylene glycol) methyl ether methacrylate] (PPEGMA) macromolecular chain-transfer agent via the RAFT process. The resulting in situ morphology changes to form shapes such as spheres, worms (toroids), and vesicles are systematically investigated. The properties of the core that determine whether a morphological change occurs from spheres are (i) the solubility of the core-forming monomer in water, (ii) the relationship between T g for the core-forming block and the polymerization temperature, and (iii) the hydrophobic core volume, which changes the packing parameter. These factors allow prediction of the block copolymer morphology produced during RAFT emulsion polymerization of other methacrylates such as n-butyl methacrylate (BuMA), tetrahydrofurfuryl methacrylate (THFMA) with physical properties of the homopolymer (poly(tetrahydrofurfuryl methacrylate) (PTHFMA)) between those for poly(MMA) (PMMA) and PBuMA, and 1-adamantyl methacrylate (ADMA) with low monomer solubility in water and high T g of the homopolymer (PADMA).

Quantitative Measurement of the Pressure and Shear Stress Acting on the Body of a Wheelchair User Using a Wearable Sheet-Type Sensor: A Preliminary Study.

To provide a safer sitting environment for wheelchair users, it is important to quantitatively measure the forces acting on the contact surface between the seat and the person in the wheelchair. In addition to the pressure acting on the buttocks, shear forces have received particular attention in recent years; however, measuring shear force is more difficult than measuring pressure. To obtain this measurement, a thin and flexible sensor that can be used in a natural state on a wheelchair is needed. Therefore, we constructed a measurement system using our previously developed wearable sheet-type sensor (0.9 mm thick). In this study, preliminary tests were conducted using human dummies before testing on humans. Sensors were placed in four locations on the humanoid dummy's back and buttocks, and the electric wheelchair was tilted and reclined five times each. The results showed that the sensor output pattern was reproducible and valid enough to proceed to the next step. However, the shear force in the internal and external directions was greater than expected, which indicates that the equipment and testing methods must be reviewed. On the basis of the results obtained in this preliminary study, preparations will be made for testing on human subjects.

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

Autonomous microfluidic control by chemically actuated micropumps and its application to chemical analyses.

Autonomous control of microfluidic transport was realized through the use of chemically actuated diaphragm micropumps connected to a network of controlling flow channels. A hydrogen peroxide (H(2)O(2)) solution was transported in the controlling flow channel by capillary action. Upon the solution's arrival at the lower compartment of a micropump filled with manganese dioxide (MnO(2)) powder, a volume change that accompanied the production of oxygen caused by the catalytic decomposition of H(2)O(2) induced inflation of the diaphragm. This in turn caused the movement of a solution in another network of flow channels formed in the upper layer. Micropumps that only exert pressure were also fabricated. By positioning the micropumps at appropriate locations in conjunction with additional flow-delaying components, the ejection of solutions from the reservoir of each micropump could be initiated at coordinated times. Furthermore, the solutions could be transported by the application of pressure from other micropumps. In other words, the information for switching from one micropump to another could be described on the chip in the form of a network of flow channels. This autonomous processing of solutions was demonstrated for enzymatic analyses of H(2)O(2), glucose, and lactate.

The relationship between torso inclination and the shearing force of the buttocks while seated in a wheelchair: Preliminary research in non-disabled individuals.

The shearing force acting upon the contacting surface of the buttocks while seated in a wheelchair has been linked to the development of pressure ulcers. However, the actual causative factors have not been examined in detail. In an attempt to clarify the nature of this problem, we developed a specific measurement system. In preparation for future clinical applications, we conducted preliminary testing in non-disabled individuals. As a result, we were able to discover a number of issues related to this system and our measurement methods. Although we only evaluated a limited number of typical sitting postures, we were able to record the buttocks shearing force reference values for non-disabled individuals. Our results suggest that regardless of posture, a backward-directed shearing force occurs below the ischial tuberosity in non-disabled individuals in seated positions. This force seemed to increase as the individual leaned forward. Meanwhile, tilting the torso to one side seemed to be associated with an increase in the shearing force working in the opposite direction. We hope that these findings can be utilized for reference purposes in future research among non-disabled individuals.

Mechanical Regulation Underlies Effects of Exercise on Serotonin-Induced Signaling in the Prefrontal Cortex Neurons.

Mechanical forces are known to be involved in various biological processes. However, it remains unclear whether brain functions are mechanically regulated under physiological conditions. Here, we demonstrate that treadmill running and passive head motion (PHM), both of which produce mechanical impact on the head, have similar effects on the hallucinogenic 5-hydroxytryptamine (5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) of rodents. PHM generates interstitial fluid movement that is estimated to exert shear stress of a few pascals on cells in the PFC. Fluid shear stress of a relevant magnitude on cultured neuronal cells induces ligand-independent internalization of 5-HT2A receptor, which is observed in mouse PFC neurons after treadmill running or PHM. Furthermore, inhibition of interstitial fluid movement by introducing polyethylene glycol hydrogel eliminates the effect of PHM on 5-HT2A receptor signaling in the PFC. Our findings indicate that neuronal cell function can be physiologically regulated by mechanical forces in the brain.

A pilot study towards long-term thermal comfort research for lower-limb prosthesis wearers.

BACKGROUND:: Thermal discomfort among lower-limb prosthesis wearers is prevalent with social and medical consequences. OBJECTIVES:: This study aimed to verify the feasibility of out-of-laboratory thermal comfort studies. STUDY DESIGN:: Repeated measures pilot study. METHODS:: Thermistors were placed on participants' residual limbs during two experimental phases. In phase 1, mean limb temperature was calculated over a controlled 55-min rest-exercise-rest protocol. In phase 2, participants conducted activities of their choosing wherever they wanted away from the lab, while limb temperature data were collected. Descriptive statistics and statistical differences between phases are presented. RESULTS:: Five male amputees participated with an average age ±standard deviation of 30 ± 9 years. In phase 1, mean limb temperature change ranged between 1.6°C and 3.7°C. In phase 2, mean limb temperature change ranged between 1.8°C and 5.1°C. Limb temperature was significantly higher in out-of-lab studies (+1.9°C, p = 0.043) compared to in-lab studies. CONCLUSION:: Independent multiple-hour temperature studies are shown to be feasible. Results also indicate that out-of-lab residual limb temperature can be significantly higher than in-lab temperatures. CLINICAL RELEVANCE: Thermal discomfort and sweating may lead to skin conditions and reduce quality of life among prosthesis wearers. Out-of-lab, long-term temperature studies are needed to comprehensively characterize thermal discomfort to create preventive solutions.

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Euodionosides A-G: megastigmane glucosides from leaves of Euodia meliaefolia.

From a 1-BuOH-soluble fraction of the MeOH extract of leaves of Euodia meliaefolia, collected in Okinawa, seven megastigmane glucosides, named euodionosides A-G, were isolated together with three known megastgmane glucosides, and two aliphatic and three phenolic compounds. Their structures were elucidated through a combination of spectroscopic analyses and application of the modified Mosher's method.

Knee extensor strength assessed using a vertical squat and a simple geometric model to calculate joint torque: An evaluation of validity and clinical utility.

AIM: We propose a simple method to measure knee extensor muscle strength using a single-legged vertical squat. The purpose of the present study was to assess the validity of this method in comparison with standard methods of measurement. METHODS: A total of 30 healthy adults (mean age 22 years) and 28 healthy older individuals (mean age 69 years) participated in this study. Knee extensor torque at maximum knee flexion during a single-leg squat was calculated by a geometrical model using height, weight and measured leg forward lean angle. Its validity as a measurement of knee extensor strength was assessed by comparing the results with maximum isometric knee extensor strength measured using an isokinetic dynamometer and a handheld dynamometer. RESULTS: Knee extensor torques calculated by the squat model were highly correlated with those calculated by the isokinetic dynamometer (r = 0.835) and handheld dynamometer (r = 0.884); however, the difference between the squat model and the isokinetic dynamometer measurements, and between the squat model and the handheld dynamometer measurements increased proportionally with increasing muscle strength. CONCLUSION: The squat model is a convenient way to measure knee extensor muscle strength quantitatively. However, it might be more suitable for individuals with quadriceps muscle weakness than for those with strong muscles. Geriatr Gerontol Int 2018; 18: 1125-1131.

Construction of a soft wearable body cooling system for persons with spinal cord injury.

This paper describes the construction of a body cooling system to avoid heatstroke for survivors of cervical spinal cord injury. For accomplishment of this purpose, we chose the neck as a cooling point of the body, and we constructed a prototype neck cooling head with a refrigerated circulator. The neck cooling head was made by thin heat-welding thermoplastic films with high thermal conductivity. To test our proposed system, we conducted experiments on two unimpaired participants in a room which simulated a hot summer day (33 [°C], relative humidity 40%). Reduction of sweating were observed, and the average skin temperatures and the core temperature of the head with cooling increased more slowly than those without cooling. The estimated cooling power of the proposed system was about 10 [W] with 50 [W] total power consumption of the cooling head.

BMPR2 mutations found in Japanese patients with familial and sporadic primary pulmonary hypertension.

Primary pulmonary hypertension (PPH) is a potentially lethal disorder, in which heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR2) gene (BMPR2) have been identified. We conducted a molecular study of BMPR2 mutations in 4 Japanese families with familial PPH and 30 Japanese patients with sporadic PPH, and found 13 different mutations, of which 10 were novel, including missense (n=2), nonsense (n=4), frameshift (n=3), and splice-donor site (n=1) mutations. In total, BMPR2 mutations were found in all 4 familial PPH cases and 12 (40%) of the sporadic PPH cases. Further, a majority of the mutations found were predicted to cause premature termination, as previously reported. In the 9 mutations found in the sporadic cases, 2 were shown to be de novo, 2 were shared in multiple cases, 1 was shared with an FPPH case, and 1 was the same as previously reported in Caucasian FPPH. These results indicate that a substantial portion of Japanese PPH patients carry BMPR2 mutations with considerable heterogeneity.

p53 protein transduction therapy: successful targeting and inhibition of the growth of the bladder cancer cells.

INTRODUCTION: Virus-mediated gene therapy for bladder cancer has some problems, such as efficiency of gene delivery and safety issues. We have reported that poly-arginine peptide (11R) has the ability to increase protein transduction in cells. Here, we show that p53 protein transduction using 11R is useful for targeting to bladder tumors and suppressing the growth of bladder cancer cells. MATERIALS AND METHODS: An 11R-fused p53 protein (11R-p53) was transduced in bladder cancer cell lines (J82 and T24) to evaluate the anti-tumor effect. Cell viability was assessed by performing the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay. To investigate whether 11R-p53 enhanced the effect on anti-cancer drug-dependent apoptosis of bladder cancer cells, the cell lines were cotreated with 11R-p53 and cis-diaminedichloroplatinum (CDDP). Apoptotic cells were identified using Hoechst staining. To investigate the efficiency of protein transduction mediated by 11R in bladder tumors in vivo, SCID mice were transplanted with J82 cells in the bladder and 11R-GFP was transurethrally transduced into the bladder. The transduction of 11R-GFP in the tumor was examined by confocal microscopy. RESULTS: 11R-p53 inhibited the growth of both J82 and T24 cells in a dose-dependent manner. The transduction of 11R-p53 enhanced CDDP-dependent induction of apoptosis. Transurethral application of 11R-GFP resulted in transduction of GFP in bladder tumors but not in the normal bladder epithelium or subepithelial tissues. CONCLUSION: The present results suggest that p53 protein transduction therapy may be a promising method for the treatment of bladder cancer.