Influence of storage condition on exosome recovery

Most of mammalian cells release extracellular vesicles including exosomes which mediate intercellular communication by delivering a variety of molecules. Despite of their importance in normal physiology and disease progression, the standard criteria of storage condition is indefinite and controversial. Therefore, we investigated exosome’s recovery yield and stability by various storage conditions. To investigate the effect of short-term storage temperature on exosome stability, exosomes were incubated at temperatures ranging from -70 to 90°C for 30 min. Immunoblot results showed that all exosome-associated proteins incubated at 90°C were mostly degraded for a short period of time. To examine the effect of long-term storage, isolated exosomes were incubated for 10 days at from -70°C to room temperature (RT), and exosomal protein, RNA and exosome markers were examined. Protein and RNA amounts were most reduced at RT compared with -70 and 4°C. Incubation at 4°C and RT resulted in major loss of CD63, and decreasing level of HSP70 was shown at only RT. In addition, flow cytometry result showed that exosome population became more dispersed after RT incubation for 10 days compared with -70°C incubated or freshly isolated exosomes. In summary, our results indicate that different storage temperature and period influences recovery yield and morphology of exosome, and storage at below -70°C is the favorable condition for preservation of fresh exosomes for clinical application and basic researches.     

Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.     

An artificial muscle actuator for biomimetic underwater propulsors

In this paper, we introduce the analytical framework of the modeling dynamic characteristics of a soft artificial muscle actuator for aquatic propulsor applications. The artificial muscle used for this underwater application is an ionic polymer-metal composite (IPMC) which can generate bending motion in aquatic environments. The inputs of the model are the voltages applied to multiple IPMCs, and the output can be either the shape of the actuators or the thrust force generated from the interaction between dynamic actuator motions and surrounding water. In order to determine the relationship between the input voltages and the bending moments, the simplified RC model is used, and the mechanical beam theory is used for the bending motion of IPMC actuators. Also, the hydrodynamic forces exerted on an actuator as it moves relative to the surrounding medium or water are added to the equations of motion to study the effect of actuator bending on the thrust force generation. The proposed method can be used for modeling the general bending type artificial muscle actuator in a single or segmented form operating in the water. The segmented design has more flexibility in controlling the shape of the actuator when compared with the single form, especially in generating undulatory waves. Considering an inherent nature of large deformations in the IPMC actuator, a large deflection beam model has been developed and integrated with the electrical RC model and hydrodynamic forces to develop the state space model of the actuator system. The model was validated against existing experimental data.     

Regulation of PTP1B via glutathionylation of the active site cysteine 215.

The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicating that the inactivated form of the phosphatase is a glutathionyl mixed disulfide. The cysteine sulfenic derivative can easily oxidize to its irreversible sulfinic and sulfonic forms and hinder the regulatory efficiency if it is not converted to a more stable and reversible end product such as a glutathionyl derivative. Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.     

Video-Assisted Thoracic Surgery for Lung Cancer Resection: A Consensus Statement of the International Society of Minimally Invasive Cardiothoracic Surgery (ISMICS) 2007

OBJECTIVE:: The purpose of this consensus conference was to determine whether video-assisted thoracic surgery (VATS) improves clinical and resource outcomes compared with conventional thoracotomy (OPEN) in adults undergoing lobectomy for lung cancer, and to outline evidence-based recommendations for the use of VATS in performing lobectomy for lung cancer. METHODS:: Before the consensus conference, the best available evidence was reviewed in that systematic reviews, randomized trials, and nonrandomized trials were considered in descending order of validity and importance. At the consensus conference, evidence-based statements were created, and consensus processes were used to determine the ensuing recommendations. The American Heart Association/American College of Cardiology system was used to label the level of evidence and class of recommendation. RESULTS AND RECOMMENDATIONS:: The consensus panel agreed upon the following statements and recommendations in patients with clinical stage I nonsmall cell lung cancer undergoing lung lobectomy:     

Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation

Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G₁ transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G₁ population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy.     

Immunoprotective activity and safety of a respiratory syncytial virus vaccine: mucosal delivery of fusion glycoprotein with a CpG oligodeoxynucleotide adjuvant

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.     

Low affinity binding of an LFA-3/IgG1 fusion protein to CD2+ T cells is independent of cell activation

Quantitative analysis of binding of the bivalent recombinant soluble fusion protein, LFA-3/IgG1, shows that the fusion protein binds to human CD2+ PBLs primarily through low affinity (KD approximately 140 microM) but also through high avidity (90 nM) interactions. The concentration dependence for LFA-3/IgG1 PBL binding took the form of two overlapping bell-shaped curves separated by a clear and reproducible minimum. This was accounted for in part by minor heterogeneity in the LFA-3/IgG1 preparations, and potentially by the ability of the ligand to bind to both CD2 and Fc receptors (FcR), best evidenced by the distinct binding properties of the fusion protein to NK and T cells. The low affinity LFA-3/ IgG1 binding to T cells is consistent with binding to CD2 only, and is in agreement with the low affinity reported for interactions between soluble forms of LFA-3 and CD2 by surface plasmon resonance technology. Moreover, as the low affinity determinations are similar for CD2 on resting and activated T cells, although the CD2 molecule has been reported to be altered to reveal new epitopes upon T cell activation, the binding data argue against multiple cell activation-dependent affinity states of CD2 for LFA-3 binding. This is distinct from that observed with other adhesion partners, and suggests that the different adhesion pathways utilize distinct mechanisms to mediate cell adhesion.