Piezo1 activation induces fibronectin reduction and PGF2α secretion via arachidonic acid cascade.

Glaucoma is a neurodegenerative disease that leads to blindness, and lowering intraocular pressure (IOP) is very important in glaucoma treatment. The trabecular meshwork is responsible for aqueous humor outflow, and the accumulation of fibronectin in trabecular meshwork is known to cause ocular hypertension. We have already shown that Piezo1 activation has an IOP lowering effect in mice and suppresses fibronectin expression level in human trabecular meshwork cells (HTMC). In this study, we report the mechanism of the reduction of fibronectin caused by Piezo1 activation. Activation of Piezo1 in HTMC showed increased expression of matrix metalloproteinase-2 (MMP-2) and cyclooxygenase (COX)-2, and decreased fibronectin expression. In addition, Piezo1 activation enhanced phosphorylation of cytosolic phospholipase A2 (cPLA2), and inhibitors targeting cPLA2 and COX-2 suppressed Yoda 1, a Piezo1 agonist, induced fibronectin reduction. These results indicate that the arachidonic acid cascade underlies this reaction, and, in support of this hypothesis, activation of Piezo1 promoted secretion of prostaglandin F2α (PGF2α) in HTMC. These results indicate that the activation of Piezo1 in HTMC promotes the degrading of fibronectin by promoting the arachidonic acid cascade and increasing the expression of PGF2α and MMP-2.

Cutaneous papilloma and multicentric squamous cell carcinoma in four-toed hedgehogs (Atelerix albiventris).

Skin lesions possibly caused by Papillomavirus infections in two four-toed hedgehogs are described. In case 1, there was a papillary mass on the right hind limb. Histologically, the mass was consistent with a viral papilloma. In the other case, multifocal papillary masses with erosions and ulcers were found throughout the body, mainly on the extremities. Histology showed continuative lesions composed of acanthosis, Bowenoid in situ carcinoma, and squamous cell carcinoma, with abrupt transitions between the lesions. In both cases, keratinocytes in the granular layer infrequently had features of koilocytes and intranuclear inclusion bodies, and immunohistochemical staining was positive for anti-human papillomavirus antibody. To the best of the authors' knowledge, this is the first pathological documentation of possibly papillomavirus-associated skin lesions in four-toed hedgehogs.

Involvement of endoplasmic reticulum stress in rotenone-induced leber hereditary optic neuropathy model and the discovery of new therapeutic agents.

Leber hereditary optic neuropathy (LHON) is caused by mitochondrial DNA mutations and is the most common inherited mitochondrial disease. It is responsible for central vision loss in young adulthood. However, the precise mechanisms of onset are unknown. This study aimed to elucidate the mechanisms underlying LHON pathology and to discover new therapeutic agents. First, we assessed whether rotenone, a mitochondrial complex Ⅰ inhibitor, induced retinal degeneration such as that in LHON in a mouse model. Rotenone decreased the thickness of the inner retina and increased the expression levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and immunoglobulin heavy-chain binding protein (BiP). Second, we assessed whether rotenone reproduces LHON pathologies on RGC-5, a neural progenitor cell derived from the retina. Rotenone increased the cell death rate, ROS production and the expression levels of ER stress markers. During chemical compounds screening, we used anti-oxidative compounds, ER stress inhibitors and anti-inflammatory compounds in a rotenone-induced in vitro model. We found that SUN N8075, an ER stress inhibitor, reduced mitochondrial ROS production and improved the mitochondrial membrane potential. Consequently, the ER stress response is strongly related to the pathologies of LHON, and ER stress inhibitors may have a protective effect against LHON.

Piezo 1 is involved in intraocular pressure regulation.

Trabecular meshwork (TM) regulates the intraocular pressure (IOP) through the control of aqueous humor outflow. Previous reports show that TM cells express 11 types of mechanosensitive molecules, including Piezo 1, which sense mechanical stimuli. However, the role of Piezo 1 on TM remains unclear. Thus, in this study, we focused on the Piezo 1 and examined its role in TM cells. Immunostaining showed that Piezo 1 was expressed in mouse TM and human TM cells. Moreover, the eye drops containing Piezo 1 agonist Yoda 1 reduced the IOP in mice, and also reduced fibronectin expression level around the TM. In addition, Piezo 1 activation suppressed human TM cells migration/proliferation, and decreased fibronectin expression level. On the other hand, Piezo 1 activation increased matrix metalloproteinase (MMP)-2 expression responsible for fibronectin degradation. These findings could contribute to the development of new treatments for glaucoma.

High Accuracy Heartbeat Detection from CW-Doppler Radar Using Singular Value Decomposition and Matched Filter.

Heart rate measurement using a continuous wave Doppler radar sensor (CW-DRS) has been applied to cases where non-contact detection is required, such as the monitoring of vital signs in home healthcare. However, as a CW-DRS measures the speed of movement of the chest surface, which comprises cardiac and respiratory signals by body motion, extracting cardiac information from the superimposed signal is difficult. Therefore, it is challenging to extract cardiac information from superimposed signals. Herein, we propose a novel method based on a matched filter to solve this problem. The method comprises two processes: adaptive generation of a template via singular value decomposition of a trajectory matrix formed from the measurement signals, and reconstruction by convolution of the generated template and measurement signals. The method is validated using a dataset obtained in two different experiments, i.e., experiments involving supine and seated subject postures. Absolute errors in heart rate and standard deviation of heartbeat interval with references were calculated as 1.93±1.76bpm and 57.0±28.1s for the lying posture, and 9.72±7.86bpm and 81.3±24.3s for the sitting posture.

Tracheal chondrosarcoma in a blue tegu (Salvator merianae).

An 8-year-and-1-month-old female blue tegu (Salvator merianae) was brought to a clinician with severe cough. The patient died 11 days later despite supportive care, and necropsy was performed by a clinician. The lumen of the distal trachea was almost completely occluded by a milk-white, hard mass measuring 1 cm in diameter. Histopathologically, the mass comprised spindle-shaped to polygonal cells arranged in a loose sheet with abundant production of basophilic cartilaginous matrix, consistent with chondrosarcoma. Reports of tracheal neoplasms in reptiles are limited, and to the best of our knowledge this represents the first pathological documentation of tracheal chondrosarcoma in a reptile.

Treatment with GDF15, a TGFß superfamily protein, induces protective effect on retinal ganglion cells.

Growth differentiation factor 15 (GDF15) is a protein belonging to the transforming growth factor beta (TGF-ß) superfamily. The precursor GDF15 is cleaved and activated as a mature GDF15 by protease. GDF15 has been detected in the aqueous humor of the primary open angle glaucoma patients, however the localization and the effect on the retinal ganglion cells (RGCs) are still unknown. Thus, the purpose of this study was to elucidate the effect of GDF15 on mouse optic nerve crush (ONC) model and primary culture of rat RGCs. Immunostaining showed that the GDF15 was in the ganglion cell layer (GCL), and colocalized with GFAP-positive cells in the GCL and the optic nerve. Western blotting analysis showed that the mature GDF15 was upregulated in the retina and the optic nerve after the ONC. Intravitreal injection of GDF15 suppressed RGCs loss of the ONC model mice in vivo. The neurites length of the primary culture of rat RGCs were increased by mature GDF15 treatment. In addition, the neurotrophic effect of GDF15 was canceled by RET inhibitor treatment. These findings indicate that GDF15 has neuroprotective effect on RGCs via GFRAL-RET pathway. Therefore, GDF15 may be one of novel therapeutic targets in RGC degenerative diseases.

Contactless Heartbeat Detection from CW-Doppler Radar using Windowed-Singular Spectrum Analysis.

The continuous-wave Doppler radar measures the movement of a chest surface including of cardiac and breathing signals and the body movement. The challenges associated with extracting cardiac information in the presence of respiration and body movement have not been addressed thus far. This paper presents a novel method based on the windowed-singular spectrum analysis (WSSA) for solving this issue. The algorithm consists of two processes: signal decomposition via WSSA followed by the reconstruction of decomposed heartbeat signals through convolution. An experiment was conducted to collect chest signals in 212 people by Doppler radar. In order to confirm the effect of reducing the large noise by the proposed method, we evaluated 136 signals that were considered to contain respiration body movements from the collected signals. When comparing to the performance of a band-pass filter, the proposed analysis achieves improved beat count accuracy. The results indicate its applicability to contactless heartbeat estimation under involving respiration and body movements.

Piezo channel plays a part in retinal ganglion cell damage.

Piezo channel is one of the mechanosensitive channels that senses pressure and shearing stress. Previous reports show that Piezo channel is expressed in many tissues such as skin and lung and they have many important roles. In addition, the mRNA of Piezo has been detected in astrocytes in the optic nerve head of mice. However, it is not yet clear where Piezo channel localize in eye and what kind of effects it have. Thus, the purpose of this study was to determine the expression sites of Piezo channel in mouse eyes and effect of Piezo channel on retinal ganglion cells. Immunostaining analysis showed that the Piezo 1/2 were expressed in the cornea, trabecular meshwork of the anterior ocular segment, lens epithelial cells, and on the retinal ganglion cell layer. The expression of retinal Piezo 2 was increased in retinal disorder model mouse caused by high IOP. Piezo 1 agonist Yoda 1 suppressed neurite outgrowth in retinal ganglion cells. On the other hand, Piezo antagonist GsMTx4 promoted neurite outgrowth in retinal ganglion cells. These findings indicate that Piezo channel may contribute to diseases relating the IOP such as glaucoma.

Energy exchange network of inter-residue interactions within a thermally fluctuating protein molecule: A computational study.

Protein function is regulated not only by the structure but also by physical dynamics and thermal fluctuations. We have developed the computer program, CURrent calculation for proteins (CURP), for the flow analysis of physical quantities within thermally fluctuating protein media. The CURP program was used to calculate the energy flow within the third PDZ domain of the neuronal protein PSD-95, and the results were used to illustrate the energy exchange network of inter-residue interactions based on atomistic molecular dynamics simulations. The removal of the α3 helix is known to decrease ligand affinity by 21-fold without changing the overall protein structure; nevertheless, we demonstrated that the helix constitutes an essential part of the network graph.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs.

Structure and mutation analysis of archaeal geranylgeranyl reductase.

The crystal structure of geranylgeranyl reductase (GGR) from Sulfolobus acidocaldarius was determined in order to elucidate the molecular mechanism of the catalytic reaction. The enzyme is a flavoprotein and is involved in saturation of the double bonds on the isoprenoid moiety of archaeal membranes. The structure determined in this study belongs to the p-hydroxybenzoate hydroxylase family in the glutathione reductase superfamily. GGR functions as a monomer and is divided into the FAD-binding, catalytic and C-terminal domains. The catalytic domain has a large cavity surrounded by a characteristic YxWxFPx(7-8)GxG motif and by the isoalloxazine ring of an FAD molecule. The cavity holds a lipid molecule, which is probably derived from Escherichia coli cells used for over-expression. One of the two forms of the structure clarifies the presence of an anion pocket holding a pyrophosphate molecule, which might anchor the phosphate head of the natural ligands. Mutational analysis supports the suggestion that the three aromatic residues of the YxWxFPx(7-8)GxG motif hold the ligand in the appropriate position for reduction. Cys47, which is widely conserved in GGRs, is located at the si-side of the isoalloxazine ring of FAD and is shown by mutational analysis to be involved in catalysis. The catalytic cycle, including the FAD reducing factor binding site, is proposed on the basis of the detailed analysis of the structure.

Development of a compact system for the determination of lead using a liquid core waveguide and a blue diode as a light source.

A simple and compact system was developed for the on-site analysis of lead in environmental water samples. The system consisted of a custom-made blue diode as a light source, a liquid core waveguide (LCW) as spectrophotometric cell and a compact UV-visible spectrometer. It weighed within 1.5 kg in total. Lead was detected using a spectrophotometric indicator reagent (TPPS: 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate) after masking interfering ions by coprecipitation with magnesium hydroxide. With an increase in the length of the LCW cell, the detection limit of lead was almost linearly enhanced, and the linear calibration range was shifted to a lower concentration range. The detection limit of lead by using 100 cm of a LCW cell was 2 nmol dm(-3). The observed value (9.6 +/- 0.4 microg dm(-3)) of lead in a river-water reference material (JSAC 0302-3) with this system was in good agreement with the certified value (9.9 +/- 0.2 microg dm(-3)).

Efficient stereoselective synthesis of gamma-N-glycosyl asparagines by N-glycosylation of primary amide groups.

The efficient and elegant synthesis of N-glycosides by N-glycosylation of asparagine-containing peptides is described. Glycosylation of primary amides with glycosyl N-phenyltrifluoroimidates in the presence of a catalytic amount of TMSOTf in nitromethane smoothly proceeded to provide the corresponding N-glycosyl amino acids in excellent yields. This coupling method was adaptable to the coupling of various glycosyl donors with amino acids and peptides.

A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.

Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.