Phosphoinositide-dependent regulation of VAN3 ARF-GAP localization and activity essential for vascular tissue continuity in plants

ACAP-type ARF GTPase activating proteins (ARF-GAPs) regulate multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion and cell migration. However, the regulation of ACAP functions by other cellular proteins is poorly understood. We have reported previously that a plant ACAP, VAN3, plays a pivotal role in plant venation continuity. Here, we report on newly identified VAN3 regulators: the CVP2 (cotyledon vascular pattern 2) 5 PTase, which is considered to degrade IP3 and also to produce PtdIns(4) P from PtdIns(4,5) P-2; and a PH domain-containing protein, VAB (VAN3 binding protein). Combinational mutations of both CVP2 and its closest homologue CVL1 (CVP2 like 1) phenocopied the strong allele of van3 mutants, showing severe vascular continuity. The phenotype of double mutants between van3, cvp2 and vab suggested that VAN3, CVP2 and VAB function in vascular pattern formation in the same pathway. Localization analysis revealed that both CVP2 and VAB colocalize with VAN3 in the trans-Golgi network (TGN), supporting their functions in the same pathway. The subcellular localization of VAN3 was dependent on its PH domain, and mislocalization of VAN3 was induced in cvp2 or vab mutants. These results suggest that CVP2 and VAB cooperatively regulate the subcellular localization of VAN3 through the interaction between its PH domain and phosphoinositides and/or inositol phosphates. In addition, PtdIns(4) P, to which VAN3 binds preferentially, enhanced the ARF-GAP activity of VAN3, whereas IP3 inhibited it. These results suggest the existence of PtdIns(4) P and/or IP3-dependent subcellular targeting and regulation of VAN3 ACAP activity that governs plant vascular tissue continuity

Scintigraphic studies on the etiology of Ampulla Cardiomyopathy

SummaryBackgroundAlthough there are many reports on Ampulla Cardiomyopathy, its etiologic mechanisms are not well known.AimEtiology of Ampulla Cardiomyopathy was investigated by myocardial scintigraphy with various nuclear tracers.Subjects and methodsIn nine patients with Ampulla Cardiomyopathy, myocardial scintigraphy was performed at acute, subacute and chronic phases. Total defect score (TDS) of tallium-201 (Tl) or technetrium-99m sestamibi (MIBI) myocardial perfusion and iodine-123-beta-methyl-p-iodophenyl penta-decanoic acid (BMIPP) scintigraphies was calculated. Cardio-mediastinal ratio (H/M) and washout rate (WR) of early and delayed images of iodine-123-meta-iodobenzylguanidine (MIBG) scintigraphy were also calculated. The patients in whom TDS of myocardial perfusion scintigraphy at acute phase was 0, were classified into group N (n=5) and those with TDS≥1 into group D (n=4).ResultsTDS of BMIPP at acute, subacute and chronic phases was higher in D than in N; 28.8±10.3 vs. 7.2±4.7 (p=0.0039), 15.5±2.1 vs. 1.0±0.8 (p<0.0001) and 2.7±1.2 vs. 0 (p=0.05), respectively. WR of MIBG at acute phase was also higher in D (50.3±5.7% vs. 36.6±10.5%, p=0.05). H/M (dH/M) on the delayed images and WR at chronic phase were not different between the two groups. H/M (eH/M) on the early images was lower in D. Blood noradrenaline (ng/ml) at acute phase was higher in D than in N (1.21±0.55 vs. 0.45±0.33, p<0.05). Left ventricular ejection fraction (LVEF) was decreased in both at acute phase but it was lower in D than in N (48.1±3.7% vs. 69.9±9.7%, p<0.05) at subacute phase.ConclusionThese findings suggest that the etiology of Ampulla Cardiomyopathy is neurologically stunned myocardium induced by coronary microcirculatory disorder.Due to the significant amount of time that was necessary for normalization of wall motion in the D group, myocardial scintigraphy is believed to be also useful in assessment of severity

Rho-associated kinase inhibitor eye drop (Ripasudil) transiently alters the morphology of corneal endothelial cells

PURPOSE: Ripasudil (Glanatec), a selective Rho-associated coiled coil-containing protein kinase (ROCK) inhibitor, was approved in Japan in September 2014 for the treatment of glaucoma and ocular hypertension. The purpose of this study was to investigate the effect of ripasudil eye drops on corneal endothelial morphology, as ROCK signaling is known to modulate the actin cytoskeleton. METHODS: Morphological changes in the corneal endothelium were evaluated in human subjects by specular and slit-lamp microscopy, following topical administration of ripasudil. We also used a rabbit model to evaluate the effect of ripasudil on clinical parameters of the corneal endothelium. Twenty-four hours after ripasudil application, corneal specimens were evaluated by phalloidin staining, immunohistochemical analysis, and electron microscopy. RESULTS: Specular microscopy revealed morphological changes in human eyes, and slit-lamp microscopy showed guttae-like findings. The rabbit model showed morphological changes similar to those seen in human eyes after ripasudil administration. Electron microscopy demonstrated that these alterations are due to the formation of protrusions along the cell-cell borders, but this formation is transient. Expression of corneal endothelial function-related markers was not disrupted; corneal thickness and corneal volume were not changed; and no cell death was observed following ripasudil administration. CONCLUSIONS: Ripasudil induces transient guttae-like findings in humans, most likely due to protrusion formation along intracellular borders caused by the reduction in actomyosin contractility of the corneal endothelial cells. No severe adverse effects were observed. Physicians should be aware that ROCK inhibitors can cause these guttae-like findings, to avoid misdiagnosing patients as having Fuchs endothelial corneal dystrophy. (www.umin.ac.jp/ctr number, UMIN000018340.)

Nanoparticle-mediated endothelial cell-selective delivery of pitavastatin induces functional collateral arteries (therapeutic arteriogenesis) in a rabbit model of chronic hind limb ischemia

ObjectivesWe recently demonstrated in a murine model that nanoparticle-mediated delivery of pitavastatin into vascular endothelial cells effectively increased therapeutic neovascularization. For the development of a clinically applicable approach, further investigations are necessary to assess whether this novel system can induce the development of collateral arteries (arteriogenesis) in a chronic ischemia setting in larger animals.MethodsChronic hind limb ischemia was induced in rabbits. They were administered single injections of nanoparticles loaded with pitavastatin (0.05, 0.15, and 0.5 mg/kg) into ischemic muscle.ResultsTreatment with pitavastatin nanoparticles (0.5 mg/kg), but not other nanoparticles, induced angiographically visible arteriogenesis. The effects of intramuscular injections of phosphate-buffered saline, fluorescein isothiocyanate (FITC)-loaded nanoparticles, pitavastatin (0.5 mg/kg), or pitavastatin (0.5 mg/kg) nanoparticles were examined. FITC nanoparticles were detected mainly in endothelial cells of the ischemic muscles for up to 4 weeks. Treatment with pitavastatin nanoparticles, but not other treatments, induced therapeutic arteriogenesis and ameliorated exercise-induced ischemia, suggesting the development of functional collateral arteries. Pretreatment with nanoparticles loaded with vatalanib, a vascular endothelial growth factor receptor (VEGF) tyrosine kinase inhibitor, abrogated the therapeutic effects of pitavastatin nanoparticles. Separate experiments with mice deficient for VEGF receptor tyrosine kinase demonstrated a crucial role of VEGF receptor signals in the therapeutic angiogenic effects.ConclusionsThe nanotechnology platform assessed in this study (nanoparticle-mediated endothelial cell-selective delivery of pitavastatin) may be developed as a clinically feasible and promising strategy for therapeutic arteriogenesis in patients.Clinical RelevanceRestoration of tissue perfusion in patients with critical limb ischemia is a major therapeutic goal. Recent clinical trials designed to induce neovascularization by administering exogenous angiogenic growth factors or cells failed to demonstrate a decisive clinical benefit. A controlled drug delivery system for a new approach to therapeutic neovascularization therefore would be more favorable. In the present study, we applied nanoparticle-mediated delivery system and report that endothelial cell-selective delivery of pitavastatin increased the development of collateral arteries and improved exercise-induced ischemia in a rabbit model of chronic hind limb ischemia. This nanotechnology platform is a promising strategy for the treatment of patients with severe organ ischemia and represents a significant advance in therapeutic arteriogenesis over current approaches

HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse

HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28–32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24–32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals

Large-scale vortical structure detection using microphone array in a semiconductor single wafer spin cleaner

Fluctuating pressure (p’ ) of a large-scale vortical structure generated in a semiconductor single wafer spin cleaner was detected by using microphone array. Twelve microphones were installed on the exhaust cover under the rotating disk of the cleaner with their interval of 7.5° or 15°. Power spectrum densities (PSD) of p’ were compared with those of fluctuating velocity measured by PIV for various rotation angular velocities to identify fluctuations due to convection of the large-scale vortical structure. Good agreement of PSDs indicates that the large-scale structure could be detected by using microphone. Cross-correlation of p’ measured at different positions revealed that the large-scale structure convected to the downstream in the rotational direction of the disk. The convection speed was about 12 % of the angular velocity of the rotating disk. Number of the vortex in the large-scale structure was also evaluated from the time-series p’ data. Time-space contour map was made for p’ based on the data measured at the different angular position, and showed periodical swept strip patterns. Presences of the strip patterns indicate the pressure disturbances were stably convected to the downstream. From this time-space map, two-dimensional Fourier transform efficiently extracted the number of vortices in the large-scale structure

“Hook and Roll Technique” Using an Articulating Hook Cautery to Provide a Critical View during Single-incision Laparoscopic Cholecystectomy

We describe a new simple and easy technique called the "Hook and roll technique" (HRT) that uses an articulating hook cautery to provide a critical view during single incision laparoscopic cholecystectomy (SILC). A 2-cm incision is made at the umbilicus to insert three 5-mm trocars or a multichannel port. After dissection of the serosa of the dorsal and ventral sides of the gall bladder, including Calot's triangle, the angled tip of the hook cautery is inserted between the cystic artery and duct with its tip placed dorsally. The tip is then rotated in a clockwise manner to avoid bile duct injury, allowing the connective tissue between them to be hooked, coagulated and cut. This procedure is repeated several times, followed by dissection between the cystic artery and the liver bed to achieve a critical view. From December 2008 to May 2011, 121 patients underwent SILC using HRT in our hospital without any serious complications. This technique is suitable for SILC, as it is consists of simple procedures that can be performed safely and easily, even by left hand in a cross-over approach, and it allows complete dissection of Calot's triangle to achieve a critical view without using any dissector under dangerous in-line viewing

Periodical structure of vortices in a semiconductor single wafer spin cleaner

We experimentally and numerically investigated large-scale structures formed by vortices in a single wafer spin cleaner. The Q-criterion identified the vortices developed in the cleaner as the flow regions with positive second invariant of the velocity gradient tensor obtained by both the PIV and LES. The time-series two-components PIV data shows that small-vortices were clustered near and under the edge of the rotating disk and were periodically emanated from there to the housing wall of the cleaner. The emanation frequency was increased with increasing in the angular velocity of the rotating disk. Three-dimensional LES reveal that six longitudinal vortices were spirally developed from under the edge of the rotating disk to the housing wall. This structure stably rotated slower than the disk speed. Fourier analysis of the LES data agreed with that of the PIV data. This supports that the passages of the stable spiral vortices on the PIV measurement region resulted in the periodical emanation of the clustered small-vortices observed in the PIV. Such a very large-scale spiral structure will induce reattachment of contaminants on the wafer surface, and should be destructed for development of much higher efficient cleaner

A Surgical Cryoprobe for Targeted Transcorneal Freezing and Endothelial Cell Removal

PURPOSE: To examine the effects of transcorneal freezing using a new cryoprobe designed for corneal endothelial surgery. METHODS: A freezing console employing nitrous oxide as a cryogen was used to cool a series of different cryoprobe tip designs made of silver for high thermal conductivity. In vitro studies were conducted on 426 porcine corneas, followed by preliminary in vivo investigations on three rabbit corneas. RESULTS: The corneal epithelium was destroyed by transcorneal freezing, as expected; however, the epithelial basement membrane remained intact. Reproducible endothelial damage was optimally achieved using a 3.4 mm diameter cryoprobe with a concave tip profile. Stromal edema was seen in the pre-Descemet's area 24 hrs postfreeze injury, but this had been resolved by 10 days postfreeze. A normal collagen fibril structure was seen 1 month postfreeze, concurrent with endothelial cell repopulation. CONCLUSIONS: Transcorneal freezing induces transient posterior stromal edema and some residual deep stromal haze but leaves the epithelial basement membrane intact, which is likely to be important for corneal re-epithelialization. Localized destruction of the endothelial monolayer was achieved in a consistent manner with a 3.4 mm diameter/concave profile cryoprobe and represents a potentially useful approach to remove dysfunctional corneal endothelial cells from corneas with endothelial dysfunction

Spin-gap formation due to spin-Peierls instability in π-orbital-ordered NaO2

We have investigated the low-temperature magnetism of sodium superoxide (NaO2), in which spin, orbital, and lattice degrees of freedom are closely entangled. The magnetic susceptibility shows anomalies at T1 = 220 K and T2 = 190 K, which correspond well to the structural phase transition temperatures, and a sudden decrease below T3 = 34 K. At 4.2 K, the magnetization shows a clear stepwise anomaly around 30 T with a large hysteresis. In addition, the muon spin relaxation experiments indicate no magnetic phase transition down to T = 0.3 K. The inelastic neutron scattering spectrum exhibits magnetic excitation with a finite energy gap. These results confirm that the ground state of NaO2 is a spin-singlet state. To understand this ground state in NaO2, we performed Raman scattering experiments. All the Raman-active libration modes expected for the marcasite phase below T2 are observed. Furthermore, we find that several new peaks appear below T3. This directly evidences the low crystal symmetry, namely, the presence of the phase transition at T3.We conclude that the singlet ground state of NaO2 is due to the spin-Peierls instability