TRiPPing the sensors: The osmosensing pathway of Polycystin 2.

Mutations to polycystin-2 (PC2), a non-selective cation permeant transient receptor potential channel, results in polycystic kidney disease (PKD). Despite the disease relevance of PC2, the physiological agonist that activates PC2 has remained elusive. As one of the earliest symptoms in PKD is a urine concentrating deficiency, we hypothesized that shifts in osmolarity experienced by the collecting duct cells would activate PC2 and loss of PC2 would prevent osmosensing. We found that mice with inducible PC2 knocked out (KO) in renal tubules had dilute urine. Hyperosmotic stimuli induced a rise in endoplasmic reticulum (ER)-mediated cytosolic calcium which was absent in PC2 KO mice and PC2 KO cells. A pathologic point mutation that prevents ion flux through PC2 inhibited the calcium rise, pointing to the centrality of PC2 in the osmotic response. To understand how an extracellular stimulus activated ER-localized PC2, we examined microtubule-ER dynamics, and found that the osmotically induced calcium increase was preceded by microtubule destabilization. This was due to a novel interaction between PC2 and the microtubule binding protein MAP4 that tethers the microtubules to the ER. Finally, disruption of the MAP4-PC2 interaction prevented incorporation of the water channel aquaporin 2 following a hyperosmotic challenge, in part explaining the dilute urine. Our results demonstrate that MAP4-dependent microtubule stabilization of ER-resident PC2 is required for PC2 to participate in the osmosensing pathway. Moreover, osmolarity represents a bona fide physiological stimulus for ER-localized PC2 and loss of PC2 in renal epithelial cells impairs osmosensing ability and urine concentrating capacity.

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

Analysis of ultrasonic scattering in blood via a continuum approach.

Since the pioneering work by Reid et al. on measuring ultrasonic scattering in blood, this phenomenon has been extensively studied both theoretically and experimentally. The knowledge on ultrasonic scattering properties of blood is needed for the design of ultrasonic methods for measuring blood flow, and a better interpretation of ultrasonic images. The development of high frequency intravascular or intracardiac imaging devices raises the possibility of measuring blood properties, e.g., erythrocyte aggregation and fibrinogen concentration, in situ. A number of theoretical approaches have been developed to analyze this phenomenon where in general ultrasound wavelength is much greater than the erythrocytes. These results show that the backscattering coefficient of blood, defined as power backscattered by a unit volume of blood per steradian per unit incident intensity, is proportional to variance of the erythrocyte number fluctuation and backscattering cross-section of a single erythrocyte. In this paper, we will show that similar results can also be obtained by taking a continuum approach.

High frequency ultrasonic backscatter from erythrocyte suspension.

Previous studies have shown that ultrasonic backscattering from red blood cells suspended in saline is proportional to the fourth power of frequency for frequencies below 15 MHz, as predicted by Rayleigh scattering theory. Recently, we have extended the measurements up to 30 MHz, because scattering of ultrasound by red blood cells may no longer be negligible at these frequencies and can affect, to a great degree, the operation of intravascular imaging devices. The experimental results show that the fourth power dependence on frequency of the backscattering coefficient for porcine erythrocytes suspended in saline solution appears to be valid up to 30 MHz. To confirm this, backscattering cross-section of porcine red cells was computed as a function of frequency using the T-matrix method. Since at higher frequencies the shape of the scatterers may also play a significant role, its effect was investigated by treating the red cell as a sphere, a disc, and a biconcave disc of the same volume. Good agreement was obtained between the experimental and theoretical results.

Ultrasound scattering from blood with hematocrits up to 100%.

The backscattering coefficient of saline suspensions of porcine red blood cells was measured for hematocrits up to about 90%. It was found that the coefficient peaks at approximately 15%, but then, contrary to what a simple "gap theory" might suggest, it decays smoothly to zero, without showing another peak at high hematocrits. A one-dimensional (1-D) slab scattering model, in which the number of slabs per unit length represents the hematocrit and whose thickness and acoustical properties are similar to red cells/plasma, was also used to investigate the relation between the backscattered power and hematocrit. Monte-Carlo simulations performed for randomized boundary conditions show a similar relation to that of the 3-D system. The experimental data is compared to the Percus-Yevick theory for the packing of hard spheres, and the simulated data is compared to the Percus-Yevick theory for infinite slabs.

A novel method for the measurement of acoustic speed.

Traditional methods for measuring acoustic speed require knowledge of either the specimen thickness or the distances between the transducers and the specimen. In general, the accuracy in measuring these quantities determines the accuracy of the experimental technique for measuring speed. This problem is particularly acute in measuring sound speed in biological specimens. A new method for measuring acoustic speed of materials, which eliminates the need for determining these quantities, has been developed. The technique, which necessitates the use of only one transducer, requires measurement of four times of flight of a sound pulse and the knowledge of the speed of sound in a reference fluid medium in which the specimen is placed. Ultrasonic speed in stainless steel and Plexiglas was measured using this method to verify its validity. Results on measurements on porcine liver, myocardium, and soft fat are also reported.