Dynamics of bubble-bubble interactions experiencing viscoelastic drag.

The subject of the present theoretical study is the dynamics of bubble-bubble interactions in a viscoelastic medium. First, new equations for calculating the viscoelastic drag exerted on bubbles during their translational motion in a viscoelastic medium are derived. The drag equations are incorporated in the bubble-bubble interaction model in which, thereby, both the translational and radial motions of the bubbles are affected by the viscoelastic features of the medium. Second, the derived equations are applied to investigate how the viscoelastic properties of the medium can affect the dynamics of multiple bubbles, as well as how the bubbles can affect each other. It was discovered that the bubble-bubble interaction can significantly influence the dynamics of a single bubble. As the distance between the bubbles increases, their effect on each other decreases, and at a distance of several millimeters, this effect can be neglected. Moreover, it was concluded that with increasing elasticity and viscosity of the medium, as well with decreasing relaxation time, the effects of other bubbles on the current bubble's radial motion can become negligible. The translational motion of the bubbles was investigated for different viscoelastic models. The elasticity resists the motion of bubbles in space, resulting in a dynamical steady state of the distance between the bubbles at high elasticity values. The relaxation time of the medium was also found to be important in terms of the bubbles' translational movement.

Simulation of cavitation enhanced temperature elevation in a soft tissue during high-intensity focused ultrasound thermal therapy.

The present study aims to investigate temperature distribution caused by bubble oscillations in a soft tissue during focused ultrasound therapy by introducing a coupled temperature-cavitation model. The proposed model is capable of describing bubble dynamics, viscoelastic properties of surrounding tissue-like medium, temperature distribution inside and outside the bubble, vapor diffusion within the bubble and vapor flux through the bubble wall to the exterior. The continuous temperature distribution inside and outside the oscillating bubble in soft tissue subject to ultrasound wave with high acoustic pressure is presented. The temperature close to the bubble wall can reach the value of about 103 K. The elasticity of soft tissue reduces temperature values. The relaxation time effect strongly depends on the period of the ultrasound wave. If the vapor mass flux effect is taken into account in the simulations, the rectified growth effect can be observed, which can lead to the decrease of the temperature values. Due to the growth of the bubble, the effects of elasticity and relaxation time on the temperature become less prominent during several bubble oscillation cycles. The impact of cavitation heat source terms on the exterior temperature was examined and led us to draw conclusion that, even though these heat sources can increase the outside temperature values, they can not be treated as main mechanisms for the temperature elevation during a few microseconds. The performed comparison with uncoupled conventional model for the outside temperature calculation revealed that coupling with inside temperature model delivers incomparably higher values to the bubble's exterior and, therefore, it is essential for the accurate description of the treatment process.

Bubble dynamics in viscoelastic soft tissue in high-intensity focal ultrasound thermal therapy.

The present study is aimed to investigate bubble dynamics in a soft tissue, to which HIFU's continuous harmonic pulse is applied by introducing a viscoelastic cavitation model. After a comparison of some existing cavitation models, we decided to employ Gilmore-Akulichev model. This chosen cavitation model should be coupled with the Zener viscoelastic model in order to be able to simulate soft tissue features such as elasticity and relaxation time. The proposed Gilmore-Akulichev-Zener model was investigated for exploring cavitation dynamics. The parametric study led us to the conclusion that the elasticity and viscosity both damp bubble oscillations, whereas the relaxation effect depends mainly on the period of the ultrasound wave. The similar influence of elasticity, viscosity and relaxation time on the temperature inside the bubble can be observed. Cavitation heat source terms (corresponding to viscous damping and pressure wave radiated by bubble collapse) were obtained based on the proposed model to examine the cavitation significance during the treatment process. Their maximum values both overdominate the acoustic ultrasound term in HIFU applications. Elasticity was revealed to damp a certain amount of deposited heat for both cavitation terms.

Refined energy-conserving dissipative particle dynamics model with temperature-dependent properties and its application in solidification problem.

It has been observed previously that the physical behaviors of Schmidt number (Sc) and Prandtl number (Pr) of an energy-conserving dissipative particle dynamics (eDPD) fluid can be reproduced by the temperature-dependent weight function appearing in the dissipative force term. In this paper, we proposed a simple and systematic method to develop the temperature-dependent weight function in order to better reproduce the physical fluid properties. The method was then used to study a variety of phase-change problems involving solidification. The concept of the "mushy" eDPD particle was introduced in order to better capture the temperature profile in the vicinity of the solid-liquid interface, particularly for the case involving high thermal conductivity ratio. Meanwhile, a way to implement the constant temperature boundary condition at the wall was presented. The numerical solutions of one- and two-dimensional solidification problems were then compared with the analytical solutions and/or experimental results and the agreements were promising.

Decreased BMP2 signal in GIT1 knockout mice slows bone healing.

Endochondral ossification, an important stage of fracture healing, is regulated by a variety of signaling pathways. Transforming growth factor ß (TGFß) superfamily plays important roles and comprises TGFßs, bone morphogenetic proteins (BMPs), and growth differentiation factors. TGFßs primarily regulate cartilage formation and endochondral ossification. BMP2 shows diverse efficacy, from the formation of skeleton and extraskeletal organs to the osteogenesis and remodeling of bone. G-protein-coupled receptor kinase 2-interacting protein-1 (GIT1), a shuttle protein in osteoblasts, facilitates fracture healing by promoting bone formation and increasing the secretion of vascular endothelial growth factor. Our study examined whether GIT1 regulates fracture healing through the BMP2 signaling pathway and/or through the TGFß signaling pathway. GIT1 knockout (KO) mice exhibited delayed fracture healing, chondrocyte accumulation in the fracture area, and reduced staining intensity of phosphorylated Smad1/5/8 (pSmad1/5/8) and Runx2. Endochondral mineralization diminished while the staining intensity of phosphorylated Smad2/3 (pSmad2/3) showed no significant change. Bone marrow mesenchymal stem cells extracted from GIT1 KO mice showed a decline of pSmad1/5/8 levels and of pSmad1/5/8 translocated into the cell nucleus after BMP2 stimulus. We detected no significant change in the pSmad2/3 level after TGFß1 stimulus. Data obtained from reporter gene analysis of C3H10T1/2 cells cultured in vitro confirmed these findings. GIT1-siRNA inhibited transcription in the cell nucleus via pSmad1/5/8 after BMP2 stimulus but had no significant effect on transcription via pSmad2/3 after TGFß1 stimulus. Our results indicate that GIT1 regulates Smad1/5/8 phosphorylation and mediates BMP2 regulation of Runx2 expression, thus affecting endochondral ossification at the fracture site.

BMP-2 induces ATF4 phosphorylation in chondrocytes through a COX-2/PGE2 dependent signaling pathway.

OBJECTIVE: Bone morphogenic protein (BMP)-2 is approved for fracture non-union and spine fusion. We aimed to further dissect its downstream signaling events in chondrocytes with the ultimate goal to develop novel therapeutics that can mimic BMP-2 effect but have less complications. METHODS: BMP-2 effect on cyclooxygenase (COX)-2 expression was examined using Real time quantitative PCR (RT-PCR) and Western blot analysis. Genetic approach was used to identify the signaling pathway mediating the BMP-2 effect. Similarly, the pathway transducing the PGE2 effect on ATF4 was investigated. Immunoprecipitation (IP) was performed to assess the complex formation after PGE2 binding. RESULTS: BMP-2 increased COX-2 expression in primary mouse costosternal chondrocytes (PMCSC). The results from the C9 Tet-off system demonstrated that endogenous BMP-2 also upregulated COX-2 expression. Genetic approaches using PMCSC from ALK2(fx/fx), ALK3(fx/fx), ALK6(-/-), and Smad1(fx/fx) mice established that BMP-2 regulated COX-2 through activation of ALK3-Smad1 signaling. PGE-2 EIA showed that BMP-2 increased PGE2 production in PMCSC. ATF4 is a transcription factor that regulates bone formation. While PGE2 did not have significant effect on ATF4 expression, it induced ATF4 phosphorylation. In addition to stimulating COX-2 expression, BMP-2 also induced phosphorylation of ATF4. Using COX-2 deficient chondrocytes, we demonstrated that the BMP-2 effect on ATF4 was COX-2-dependent. Tibial fracture samples from COX-2(-/-) mice showed reduced phospho-ATF4 immunoreactivity compared to wild type (WT) ones. PGE2 mediated ATF4 phosphorylation involved signaling primarily through the EP2 and EP4 receptors and PGE2 induced an EP4-ERK1/2-RSK2 complex formation. CONCLUSIONS: BMP-2 regulates COX-2 expression through ALK3-Smad1 signaling, and PGE2 induces ATF4 phosphorylation via EP4-ERK1/2-RSK2 axis.

Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway.

BACKGROUND AND OBJECTIVE: While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. MATERIAL AND METHODS: To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. RESULTS: In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. CONCLUSION: Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

IGF-1 regulation of type II collagen and MMP-13 expression in rat endplate chondrocytes via distinct signaling pathways.

OBJECTIVE: Abnormal maturation and ossification of the endplate chondrocytes play a central role in the pathogenesis of degenerative disorders of the cervical spine. It is widely held that insulin like growth factor-1 (IGF-1) stimulates chondrocyte proliferation and inhibits chondrocyte terminal differentiation both in vitro and in vivo. However, the mechanism underlying such regulation is not fully understood. The present study aimed to determine the role of IGF-1 on the mRNA expression of collagen type II, alpha 1 (Col2a1) and matrix metallopeptidase 13 (MMP-13) in rat endplate chondrocytes. The possible pathways that transduce IGF-1 effects such as phosphatidylinositol-3 (PI-3)-kinase (PI3K) and mitogen activated protein kinase (MAPK) were also investigated in these cells. METHODS: Cultured endplate chondrocytes harvested from rat cervical spines were treated with IGF-1 (100ng/ml), and the changes in Col2a1 and MMP-13 mRNA were monitored with real-time polymerase chain reaction (PCR). MMP-13 activity was also assayed. Activation of signaling proteins was evaluated by western blot analysis. Cells were also treated with pharmacological agents that block PI3K and MAPK signaling pathways. RESULTS: IGF-1 increased Col2a1 mRNA expression in rat endplate chondrocytes in a time- and dose-dependent manner. IGF-1 treatment resulted in a fourfold increase of Col2a1 mRNA with the effect maximizing at 24h. In contrast, IGF-1 treatment for 24h caused a roughly 50% reduction in MMP-13 mRNA. Similar effects were seen on the protein levels of type II collagen (col2) and MMP-13. Consistent with these results, IGF-1 also repressed MMP-13 activity. IGF-1 activated both the PI3K and the extracellular signal-regulated kinase (ERK) pathways as evidenced by phosphorylation of either Akt or ERK1/2 (respectively). The PI3K inhibitor Wartmannin significantly inhibited the IGF-1 effect on Col2a1 mRNA expression but did not affect IGF-1-induced repression of MMP-13 expression. In contrast, the ERK/MAPK inhibitor PD98059 significantly inhibited the effect of IGF-1 on MMP-13 mRNA repression and enhanced IGF-1-induced Col2a1 mRNA expression. CONCLUSIONS: In rat endplate chondrocytes the PI3K pathway mainly transduces IGF-1 effect on col2 expression while the ERK pathway mediates IGF-1 effect on MMP-13 expression.

Optimization of foot-and-mouth disease vaccination protocols by surveillance of neutralization antibodies.

An appropriate immunization program for pigs in a foot-and-mouth disease (FMD) endemic area was proposed based on data analysis obtained from serological surveillance in Taiwan, after an intensive vaccination program. To provide an adequate passive immunity for piglets, gilts that have completed two basic vaccinations must be boosted once before breeding. To achieve an efficient response to the FMD vaccine for piglets born to well vaccinated sows, vaccination need to be delayed until 10-12 weeks of ages for the first immunization, followed by a boost 4 weeks later.

c-IAP1 is cleaved by caspases to produce a proapoptotic C-terminal fragment.

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.

The in vivo effect of lipopolysaccharide on Na+,K(+)-ATPase catalytic (alpha) subunit isoforms in rat sciatic nerve.

The Na+,K(+)-ATPase catalytic (alpha) subunit in sciatic nerve of lipopolysaccharide (endotoxin, LPS)-treated rat was investigated. Using Western blot to determine subunit isoform polypeptide levels in rat sciatic nerve, we found a substantial reduction in alpha 1 polypeptide, but not that of alpha 2 and alpha 3 polypeptides, after the administration of LPS. Moreover, when rats were treated with polymyxin B (a LPS neutralizer) and NG-nitro-L-arginine (an inhibitor of nitric oxide (NO) synthase), the effects of LPS were reversed. These results implicate a specific marked deficit in alpha 1 subunit isoform of Na+,K(+)-ATPase in the pathogenesis of neuropathy during endotoxemia, through, at least in part, the L-arginine/NO pathway.

Mass spectrometric analysis of rat ovary and testis cytosolic glutathione S-transferases (GSTs): identification of a novel class-alpha GST, rGSTA6*, in rat testis.

Cytosolic glutathione S-transferases (GSTs) from rat ovaries and testis were purified by a combination of GSH and S-hexylglutathione affinity chromatography. The isolated GSTs were subjected to reverse-phase HPLC, electrospray MS and N-terminal peptide sequencing analysis. The major GST isoenzymes expressed in ovaries are subunits A3, A4, M1, M2 and P1. Other isoenzymes detected are subunits A1, M3 and M6*. In rat testis, the major GST isoenzymes expressed are subunits A3, M1, M2, M3, M5* and M6*. Subunits A1, A4 and P1 are expressed in lesser amounts. We could not detect post-translational modifications of any GSTs with known cDNA sequence. The molecular masses of subunits M5* and M6*, two class-Mu GSTs that have not been cloned, were determined to be 25495 and 26538 Da respectively. An N-terminally modified protein from rat testis with molecular mass 25737 Da was isolated from the S-hexylglutathione column. Results from internal peptide sequencing analysis indicate that this is a novel class-Alpha GST that has not been previously reported. We designate this protein rGSTA6*.

The in vivo effect of lipopolysaccharide on the spontaneous release of transmitter from motor nerve terminals.

1. The in vivo effect of E. coli lipopolysaccharide (LPS) on the spontaneous release of transmitter was studied in the isolated phrenic nerve-diaphragm preparation of the mouse. 2. The resting membrane potential was decreased and frequency of miniature endplate potentials (m.e.p.ps) was increased by treatment with LPS. 3. Pretreatment of diaphragms with ouabain markedly increased the frequency of m.e.p.ps in control group but not in the LPS group. 4. When mice were treated with polymyxin B (a LPS neutralizer), pentoxifylline (an inhibitor of tumor necrosis factor-alpha formation) and NG-nitro-L-arginine (an inhibitor of nitric oxide (NO) synthase) the effects of LPS were reversed. 5. These results suggest that LPS increases the spontaneous transmitter release through, at least in part, the pathways of tumour necrosis factor-alpha and NO followed by an inhibition of the Na(+)-pump activity in the endplate area.

Changes in lymphocyte single strand breakage and liver function of workers exposed to vinyl chloride monomer.

Vinyl chloride monomer (VCM) is a suspected human carcinogen. Its metabolite, chloroethylene epoxide, is able to alkylate the DNA molecule and to produce single strand breakage (SSB). A total of 244 workers from 4 polyvinyl chloride (PVC) manufacturing factories were recruited to assess the SSB of their peripheral lymphocyte DNA. The method of alkaline unwinding and hydroxyapatite chromatography was used to detect and calculate frequencies of SSB. In addition, hepatitis B and C markers and the liver function of the workers were also examined. The worker's cumulative exposures to VCM were retrospectively constructed from the current monitoring data and each worker's job history. Multiple linear regression models were constructed to predict the worker's level of SSB and liver functions based on various exposure indices and variables, such as age, sex, smoking, drinking, and hepatitis markers. The results showed that current smoking and drinking status, and the presence of VCM exposures on the previous day were 3 major determinants of the level of SSB. Among the liver function tests, only gamma-glutamyl transpeptidase (GGT) was associated with current VCM exposures. In contrast, aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were mainly affected by the presence of hepatitis B surface antigen (HBsAg) or anti-hepatitis C virus (anti-HCV). We conclude that GGT should be considered to be included in the regular health screening of VCM workers, and that the SSB method may not be suitable for long-term monitoring of cumulative exposure because of the quick DNA repair mechanism in humans.

Improving survival of culture bacteria in frozen desserts by microentrapment.

Lactobacillus bulgaricus cells were entrapped in beads of calcium alginate and evaluated for their ability to survive freezing processes. Cells survived freezing (without agitation) in ice milk mix much better than in distilled water, and more entrapped cells survived than did cells that were not entrapped. Glycerol and mannitol were cryoprotective, but glucose was not, when each was added (6%) separately to the beads. Entrapment protected the lactobacilli in batch frozen and continuously frozen ice milk mixes. The percentage of survival for entrapped and unentrapped cells in continuously frozen ice milk approximated 90 and 40%, respectively. Lactobacilli survived better in beads with mean diameters > 30 microns than in those averaging 15 microns. Addition of entrapped lactobacilli had no measurable effect on the sensory characteristics of the ice milk.

The role of receptor-ligand endocytosis and degradation in interleukin-2 signaling and T-lymphocyte proliferation.

The specific intracellular signaling pathways for interleukin-2 (IL-2) that lead to delivery of the proliferative stimulus are currently unknown. We and others have excluded signaling pathways used by other growth factors and by the antigen-specific T-cell receptor, such as increased intracellular Ca2+ concentrations, activation of protein kinase C, or ion transport across the plasma membrane. One feature of IL-2 signaling that may be important in delivery of the proliferative stimulus is endocytosis and processing of the lymphokine receptor-ligand complex. In this study we examined these steps in receptor signaling by mouse CTLL-2 cells and human OKT3-activated T-cells using monoclonal antibodies specific for the 55 kDa alpha-subunit of the IL-2R that allow IL-2 binding but block endocytosis, and with lysosomotrophic amines that selectively inhibit receptor mediated endocytosis and/or processing of IL-2. Our results demonstrate that these inhibitors block receptor endocytosis, ligand degradation, c-fos protooncogene activation, and ultimately proliferation of the IL-2-dependent T-cell line, CTLL-2. In heterogeneous populations of activated human T cells the lysosomotrophic amines demonstrated a greater inhibition of degradation than of endocytosis. These observations support the hypothesis that IL-2/IL-2R endocytosis and ligand/receptor processing or degradation may be important steps in lymphokine signal transduction.

Characterization of variant and parental-cross-protective immunity to immunogenic variants of a murine fibrosarcoma using the local adoptive transfer assay.

The purpose of this study was to characterize the lymphocyte populations responsible for rejection of immunogenic (Imm+) tumor variants, and the cross-protective immunity engendered by Imm+ variants against the weakly immunogenic parental tumor. Immunogenic clones of the weakly immunogenic methylcholanthrene-induced fibrosarcoma MCA-F have been generated using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation (UV-B; 280-320 nm). These clones grow progressively in immunosuppressed adult-thymectomized irradiated mice, but are rejected by immunocompetent syngeneic hosts. The parental MCA-F tumor grows progressively in both groups. Mice that have rejected a challenge of 1 x 10(5) Imm+ cells show an anamnestic immune response against both the Imm+ clone and the parental MCA-F tumor. Using the local adoptive transfer assay and depletion of T-cell subsets with antibody plus complement, we show that immunity induced by the Imm+ variants against the parent MCA-F was mediated by the Thy1.2+, L3T4a+ population without an apparent contribution by Lyt2.1+ cells. Although antivariant immunity was also dependent upon Thy1.2+ cells, depletion of either the L3T4a+ or the Lyt2.1+ cells failed to abolish immunity against the variant. A role for Lyt2.1+ T lymphocytes in antivariant immunity, but not antiparent immunity, was supported by the results of cytotoxic T lymphocyte (CTL) assays. Following immunization with high numbers (1 x 10(5) to 5 x 10(5) of viable Imm+ cells, antivariant, but not antiparent CTL activity was detected in mixed lymphocyte tumor cell cultures. Immunization with lower numbers (3 x 10(4] of viable Imm+ or with high numbers of mitomycin-C-treated Imm+ engenders only antivariant immunity without parental cross-protection. Under these conditions lymphocytes mediating immunity against the variant in the local adoptive transfer assay were exclusively of the Thy1.2+, L3T4a+ phenotype, with no contribution from the Lyt2.1+ cells. Identical results were obtained for Imm+ clones of MCA-F induced by methylnitronitrosoguanidine, 5-azadeoxycytidine, and UV-B, suggesting that the nature of the antitumor immunity engendered by Imm+ is not significantly affected by the agent used. Furthermore, these results demonstrate that the cross-reactivity and cellular effectors of antitumor immunity in this system are influenced by the immunizing dose of Imm+ cells: the predominant effectors of both antivariant and parental-cross-reactive immunity were of the CD4+ T cell subclass, with a CD8+ cytotoxic population contributing to antivariant immunity only after high-dose immunization.

Differential extraction of tumor-specific antigens from two ultraviolet light-induced murine fibrosarcomas with the use of 1-butanol.

The purpose of this study was to investigate the immunobiological characteristics of the tumor-specific cell surface antigen expressed by the UV-induced murine fibrosarcoma, UV-2240. UV-2240 is classified as a regressor UV tumor because it is immunologically rejected by normal syngeneic mice but grows in immunocompromised or UV-irradiated hosts. The strong tumor-specific rejection antigen expressed by UV-2240 was found on the plasma membrane, and unlike the previously characterized antigen of UV-1591, the UV-2240 antigen was removed by using the noncytolytic butanol extraction technique. The tumor antigen activity in butanol extracts was resistant to digestion by endoglycosidase F and alpha-mannosidase, but was destroyed by pronase. In addition, the immunoprotective activity in extracts of UV-2240 was thermostable. These data demonstrate that the UV-2240-specific tumor antigen possesses physicochemical properties distinct from those of its well-characterized counterpart UV-1591.