Real-time freehand 3D ultrasound imaging

<p>Real-time freehand 3D ultrasound (US) has attracted much attention in clinical practices because it provides interactive feedback to help the clinicians acquire not only high-quality images but also timely information of the scanning area, which is necessary in intraoperative examinations. In this study, we developed a real-time freehand 3D US imaging system which can obtain volume reconstruction and visualisation during data acquisition at real-time level. Based on two popular algorithms, i.e. squared distance weighted interpolation (SDW) and Bezier interpolation, we designed corresponding parallel computing methods that were implemented on the graphics processing unit (GPU) to incrementally reconstruct and display the tissues being scanned using Visualisation toolkits (VTK). With a typical B-scan image size of 302 × 268 at an acquisition rate of 25 Hz and a preset volume size of 90 × 81 × 192, the system achieved an incremental reconstruction-visualisation rate of up to 32 frames/s and 119 frames/s for the SDW and Bezier algorithms, respectively, achieving the real-time 3D US.</p

Influenza infection increases intracellular calcium, induces phosphorylation of MLCK, MYPT, and MLC, and activates PKC, RhoA, and Rho kinase.

<p>HUVECs were infected with influenza virus (MOI, 10) and were collected at the indicated times points [after infection] for calcium measurement by using fluo-3/Fura. Red fluorescence ratios (A), blotting for MLCK phosphorylation (B), and measurement of activities of PKC (C), RhoA (D) and Rho kinase (E). Influenza infection increases MYPT and MLC phosphorylation. HUVECs were infected with influenza virus (MOI, 10) and after the indicated time were lysed and used for MYPT-P (F) and MLC-P (G) blotting. N = 4 for each experiments.</p

Inhibition of ERK and MLC phosphorylation and actin polymerization leads to nuclear retention of influenza RNP complexes.

<p>The effect of ERK on the nuclear export of influenza RNP complexes is mediated by MLC phosphorylation. (A) HUVECs cells were cultured on chamber slides, transduced with the indicated adenovirus, and, 24 laters, transfected with the indicated siRNA. After 24 h, the cells were infected with influenza virus (MOI 10). Calyculin A (0.5 nM) was added to the indicated sample 30 minutes before the infecton. Eight h later, the cells were stained for the presence of influenza nucleoprotein (NP) protein (green). Inhibition of RhoA leads to inhibition of the nuclear export of influenza RNP complexes. (B, C) HUVECs were transduced with the indicated adenoviruses (B) and constructs (C) and were processed as described above. Polymerized actin is necessary for the nuclear export of influenza RNP complexes. (D) MDCK cells were infected with influenza virus (MOI 10) and treated 2 h later with DMSO or 30 µM of cytochalasin D. After 6 h, the cells were fixed and stained. Hoechst 33342 was used as nuclear counterstain (blue). Bar, 5 µm; N = 4 for each experiment.</p

Induction of MLC phosphorylation reverses the inhibitory effects of HRas\ERK on influenza proliferation.

<p>The anti-influenza effects of Raf kinase and MEK/ERK inhibitors were reversed when HUVECs were treated with an MLC phosphatase inhibitor. (A) MDCK cells were treated as indicated GW5074 and U0126 at 10 µM each overnight; calyculin A at 0.5 µM for 1 h] and then infected with influenza (MOI, 0.01). After 24 h, virus yield was measured in the medium by using RT-PCR. HRas-enhanced influenza proliferation is attenuated by inhibition of MLC kinase, ERK and Raf-1. (B) MDCK cells were seeded on 24-well plates, transduced with the indicated adenovirus, and transfected 24 h later with the indicated constructs. The cells were infected with influenza A virus (MOI, 0.01) and 24 h after the infection, RT-PCR was used to measure the virus yield in the medium. Inhibition of Rho kinase 1 in human bronchial epithelial cells leads to inhibition of the nuclear translocation of influenza RNP complexes. (C) Human bronchial epithelial cells were seeded on chamber slides, transfected with the indicated siRNA, and infected 24 h later with influenza virus (MOI, 10). After 8 h, the cells were stained for influenza nucleoprotein (NP) protein. Hoechst 33342 was used as a nuclear counterstain (blue). Bar, 5 µm; *P<0.01; N = 4 for each experiment.</p

HRas activation increases phosphorylation of MLC.

<p>Influenza-induced MLC phosphorylation is inhibited by inhibiting HRas, RhoA, and PKC. (A, B) HUVECs were transduced with the indicated adenoviruses and infected 48 h later with influenza virus (MOI 10). The cells were collected 8 h later for blotting. Limax flavus lectin (LFL)–induced MLC and ERK phosphorylation depends on HRas, Raf-1, PKC-α, and RhoA. (C, D) HUVECs were transduced with the indicated adenoviruses; 48 h later, the cells were treated for 15 min with LFL [100 mg/ml]. Activation of HRas, RhoA, and PKC-α leads to MLC and ERK phosphorylation. (E, F) Western blots performed 48 hours after HUVECs were transduced with empty adenovirus vector (Null) or the indicated adenoviruses. *P<0.01; N = 4 for each experiment.</p

Inhibition or induction of MLC phosphorylation inhibits or enhances influenza proliferation, respectively.

<p>Inhibition of MLCK leads to inhibition of influenza replication. MDCK cells were treated with serial dilutions of the MLC kinase inhibitor ML7 for 16 h (A) or were transfected with a DN mutant of MLCK for 24 h (B). The cells were infected with influenza virus (MOI, 0.01), and virus yield was measured 24 h later by RT-PCR. Inhibitors of Rho kinase and phospholipase C inhibitors inhibit influenza proliferation. MDCK cells were treated with the indicated concentrations of Fasudil (C) or the phospholipase C inhibitor, U73122 (D) and 16 h later were infected with influenza virus (MOI, 0.01). Treatment of HUVECs with PMA and TNF-α leads to phosphorylation of MLC. (E) HUVECs treated for 15 min with 5 nM PMA or 20 ng/ml TNF-α. Treatment of HUVECs with PMA, calyculin A or TNF-α enhances influenza proliferation. (F–H) HUVECs were treated with the indicated concentrations of PMA (F), calyculin A (G) or TNF-α (H) for 30 minutes and then infected with influenza (MOI, 0.01). After 24 h, virus yield was measured by RT-PCR. Influenza-induced MLC phosphorylation is inhibited by inhibitors of MEK/ERK, Raf kinase and phospholipase C. (I) HUVECs were pretreated with PD98059(5 Mm, 2 h), GW 5074 or U73112 (10 µM each for 2 h), infected with 10 MOI of influenza virus, and collected 8 h later. *P<0.01; N = 4 for each experiments.</p

Infection of HUVECs with influenza virus leads to remodeling of actin cytoskeleton.

<p>Treatment of HUVECs with calyculin A induces MLC phosphorylation (A). HUVECs were treated with the indicated concentration of calyculin A for 15 minutes. MLC phosphorylation leads to an increase in the F-actin to G-actin ratio (B). HUVECs were treated with 500 nM of calyculin A (caly) or DMSO (Con) and, after 15 minutes, were homogenized in F-actin stabilization buffer and then centrifuged to separate the F-actin [F] from the G-actin [G] pool. The fractions were separated by SDS-PAGE, and actin was quantified by Western blot. Influenza infection increased actin polymerization, the formation of actin stress fibers, and the assembly of myosin. HUVECs were infected with influenza (MOI, 10); 8 h later, the F/G ratio was measured (C). Actin fiber formation (D) and assembly of non-muscle myosin II (E) were assessed by specific staining using fluorescence microscopy. Actin fibers were detected by Texas-red conjugated phalloidin. Primary antibodies were used to bind myosin II, and FITC-conjugated secondardy antibodies were used for detection. Hoechst 33342 was used as nuclear counterstain (blue). Bar, 10 µm. *P<0.01. N = 4 for each experiments.</p

Validation of adenovirus transduction and siRNA and plasmid transfection.

<p>(A) The average transduction rate of HUVECs with the recombinant GFP-adenovirus was 80% to 90%. HUVECs were transduced with GFP adenovirus and after 48 h were assessed for transfection rate. Hoechst 33342 (0.5 µg/ml for 5 minutes) was used as a nuclear counter-stain. Fluorescent images were acquired by MicroSuite FIVE software (Olympus Soft Imaging Solutions) with an Olympus BX61 motorized microscope. Bar, 10 µm. (B) Transdution of HUVEC with CA-HRas and CA-Raf adenoviruses induced ERK phosphorylation. HUVECs were transduced with the indicated adenoviruses and 48 h were used for blotting. (C) Treatment of HUVECs with siRNA for MYPT, Rho kinase1/2, Gα12/13, and Gαq11 led to reduction in the mRNA expression. HUVECs were transfected with the indicated si RNA and after 24 h the m RNA leves for each gene were quatified by TaqMan real-time PCR . *P<0.01; N = 4 for each experiment. (D) Overexpression of the DN mutant of MLC kinase in HUVECs led to a 2.5-fold reduction in MLC phosphorylation. HUVECs were transfected with DN mutant of MLC kinase and 24 h later were used for blotting.</p

Pathway model used in this study.

<p>Signal transduction pathways involved in myosin light chain phosphorylation. MLC, myosin light chain; PKG, protein kinase G.</p

Engagement of sialic acid leads to phosphorylation of ERK and MLC.

<p>Treatment of HUVECs with Limax flavus lectin (LFL) leads to ERK and MLC phosphorylation (A, B). HUVECs were treated for 15 min with the indicated concentration of LFL and then lysed. G protein α12/13 mediates influenza-induced ERK phosphorylation. (C) HUVECs were tranfected with the indicated siRNA; 24 h later the cells were infected with influenza virus (MOI, 10) and then collected after 8 h. The pictures are representative of 4 independent experiments.</p