Mesenchymal Stem Cell-Derived Exosomes Mitigate Acute Murine Liver Injury via Ets-1 and Heme Oxygenase-1 Up-regulation.

BACKGROUND: Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism. METHODS: Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intraperitoneally administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment. RESULTS: Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway. CONCLUSION: Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

Synthesis of High-Input Impedance Electronically Tunable Voltage-Mode Second-Order Low-Pass, Band-Pass, and High-Pass Filters Based on LT1228 Integrated Circuits.

This paper introduces two new high-input impedance electronically tunable voltage-mode (VM) multifunction second-order architectures with band-pass (BP), low-pass (LP), and high-pass (HP) filters. Both proposed architectures have one input and five outputs, implemented employing three commercial LT1228 integrated circuits (ICs), two grounded capacitors, and five resistors. Both proposed architectures also feature one high-impedance input port and three low-impedance output ports for easy connection to other VM configurations without the need for VM buffers. The two proposed VM LT1228-based second-order multifunction filters simultaneously provide BP, LP, and HP filter transfer functions at Vo1, Vo2, and Vo3 output terminals. The pole angular frequencies and the quality factors of the two proposed VM LT1228-based second-order multifunction filters can be electronically and orthogonally adjusted by the bias currents from their corresponding commercial LT1228 ICs, and can be independently adjusted in special cases. In addition, both proposed VM LT1228-based second-order multifunction filters have two independent gain-controlled BP and LP filter transfer functions at Vo4 and Vo5 output terminals, respectively. Based on the three commercial LT1228 ICs and several passive components, simulations and experimental measurements are provided to verify the theoretical predictions and demonstrate the performance of the two proposed high-input impedance electronically tunable VM LT1228-based second-order multifunction filters. The measured input 1-dB power gain compression point (P1dB), third-order IMD (IMD3), third-order intercept (TOI) point, and spurious-free dynamic range (SFDR) of the first proposed filter were -7.1 dBm, -48.84 dBc, 4.133 dBm, and 45.02 dBc, respectively. The measured input P1dB, IMD3, TOI, and SFDR of the second proposed filter were -7 dBm, -49.65 dBc, 4.316 dBm, and 45.88 dBc, respectively. Both proposed filters use a topology synthesis method based on the VM second-order non-inverting/inverting HP filter transfer functions to generate the BP, LP and HP filter transfer functions simultaneously, making them suitable for applications in three-way crossover networks.

The Long-Term Clinical Impact of Thoracic Endovascular Aortic Repair (TEVAR) for Advanced Esophageal Cancer Invading Aorta.

BACKGROUND: Advanced esophageal cancer invading the aorta is considered unsuitable for surgery with definitive chemotherapy or chemoradiation as the treatments of choice. In the current study, we evaluated the long-term clinical impact of combining thoracic endovascular aortic repair (TEVAR) with multimodality treatment in caring for such patients. METHODS: We evaluated 48 patients who had advanced esophageal cancer with aortic invasion. The oncological outcome, including overall survival (OS) and progression-free survival (PFS), after multimodality treatment with or without TEVAR is evaluated for these patients. RESULTS: Overall, 25/48 patients (52.1%) received a TEVAR procedure. There was no significant difference in OS (p = 0.223) between patients who did or did not receive TEVAR; however, patients who received TEVAR had significantly less local tumor recurrence (p = 0.020) and longer PFS (p = 0.019). This impact was most evident in patients who received both TEVAR and esophagectomy, with an incremental increase in hazard ratio (HR) for disease progression of 2.89 (95% confidence interval [CI] 0.86-9.96) and 4.37 (95% CI 1.33-14.33) observed under multivariable analysis, respectively, in comparison with patients who underwent only one or neither of these procedures (p = 0.005 for trend test). CONCLUSION: TEVAR is a feasible procedure for esophageal cancers invading the aorta and can be used for curative-intent resection to improve local tumor control and PFS.

Infusion of Human Mesenchymal Stem Cells Improves Regenerative Niche in Thioacetamide-Injured Mouse Liver.

BACKGROUND: This study investigated whether xenotransplantation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. METHODS: Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6 weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. RESULTS: WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-ß1-stimulation in Smad2 phosphorylation and RhoA upregulation. CONCLUSION: These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

Toxic Effects of Urethane Dimethacrylate on Macrophages Through Caspase Activation, Mitochondrial Dysfunction, and Reactive Oxygen Species Generation.

Urethane dimethacrylate (UDMA) is a dimethacrylate-based resin monomer that can react with other related monomers and inorganic particles, causing hydrophobic polymerization through cross-linking upon light activation. UDMA polymers are commonly used for the reconstruction and reinforcement of teeth and bones. UDMA can become unbound and be released from light-cured polymer resins. Thus far, no evidence exists on the toxic effects of UDMA and its related working mechanisms for macrophages. Therefore, in the present study, we investigated the cytotoxicity, mode of cell death, DNA damage, caspase activities, mitochondrial dysfunction, and reactive oxygen species (ROS) generation in RAW264.7 macrophages treated with UDMA using the lactate dehydrogenase (LDH) assay kit, Annexin V-FITC and PI assays, micronucleus formation and comet assay, caspase fluorometric assay, JC-1 assay, and 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay, respectively. Our results show that UDMA induced cytotoxicity; apoptosis and necrosis; genotoxicity, which is also called DNA damage; increased caspase-3, -8, and -9 activities; mitochondrial dysfunction; and intracellular ROS generation in a concentration-dependent manner in RAW264.7 macrophages. Thus, based on the observed inhibited concentration parallel trends, we concluded that UDMA induces toxic effects in macrophages. Furthermore, UDMA-induced intracellular ROS generation, cytotoxicity, and DNA damage were reduced by N-acetyl-L-cysteine.

Propofol-enhanced autophagy increases motility and angiogenic capacity of cultured human umbilical vascular endothelial cells.

AIMS: Propofol (PPF), an intravenous anesthetic agent, is previously reported to attenuate oxidative stress- and inflammation-induced endothelial cell dysfunction. This study investigated its effect on endothelial cell biology. MAIN METHODS: Cultured human umbilical vein endothelial cells (HUVECs) were treated with PPF and subject to measurements for nitric oxide (NO) production, autophagy flux, signal transduction, migration, and in vitro angiogenesis. KEY FINDINGS: Non-cytotoxic PPF treatment was found to significantly upregulate inducible nitric oxide synthase (NOS2) but downregulate constitutive NOS3 expression. It also potentiated LPS-induced ICAM-1 overexpression and NO overproduction. Mechanistically, the PPF-activated signal transduction in PI3K/Akt, ERK1/2, p38 MAPK, and JNK pathways were involved in the PPF-driven NO overproduction. PPF exhibited a stimulatory effect on autophagy flux by increasing expression of autophagy markers including mTOR, Beclin-1, Atg5, and LC3I/II, as well as a late endosomal indicator, Rab7. However, PPF appeared to antagonize the Rab7 upregulation by LPS. Functionally, PPF enhanced in vitro migratory and angiogenic capacities of HUVECs, but this enhancement was drastically abrogated by the presence of autophagy inhibitors, indicating a pro-angiogenic contribution of PPF-enhanced autophagy in cultured HUVECs. SIGNIFICANCE: Our findings support the notion that PPF enhances motility and angiogenic capacity of cultured HUVECs through an autophagy-involved regulatory mechanism.

Toll-like receptor-4 is a target for suppression of proliferation and chemoresistance in HepG2 hepatoblastoma cells.

Toll-like receptor-4 (TLR4) is known to influence growth and migration of hepatocellular tumors; however, its role in hepatoblastoma remains poorly understood. This study investigated the regulatory role of TLR4 in proliferation and chemoresistance of HepG2 hepatoblastoma cells. Treatment with lipopolysaccharide (LPS), a TLR4 agonist, was found to significantly upregulate TLR4 expression in HepG2 cells, but not in malignant Huh-7 and Sk-Hep1 hepatocellular carcinoma cells. Additionally, IL-6 enhanced LPS-induced TLR4 upregulation. LPS-stimulated TLR4 activation increased proliferation, nitric oxide synthase (NOS) expression, and NO production in HepG2 cells. Chemotherapeutic agents, cisplatin and doxorubicin, effectively inhibited TLR4 expression in HepG2 cells. Characterization of LPS-induced signaling activation and blockade with kinase inhibitors revealed the involvement of Akt and MAPK pathways in LPS-enhanced NO release from, and proliferation of HepG2 cells. Mechanistically, gene modifications as a result of TLR4 transfection and siRNA-mediated knockdown further demonstrated a crucial role for TLR4 in the regulation of NOS expression, cell proliferation, and chemoresistance in HepG2 cells. These findings suggest that targeting TLR4 expression and its cognate signaling may modulate proliferation and chemosensitivity in hepatoblastoma cells and serve as a potential therapeutic target.

Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis.

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-ß1 (TGF-ß1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-ß1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.

Modulating malignant epithelial tumor cell adhesion, migration and mechanics with nanorod surfaces.

The failure of tumor stents used for palliative therapy is due in part to the adhesion of tumor cells to the stent surface. It is therefore desirable to develop approaches to weaken the adhesion of malignant tumor cells to surfaces. We have previously developed SiO2 coated nanorods that resist the adhesion of normal endothelial cells and fibroblasts. The adhesion mechanisms in malignant tumor cells are significantly altered from normal cells; therefore, it is unclear if nanorods can similarly resist tumor cell adhesion. In this study, we show that the morphology of tumor epithelial cells cultured on nanorods is rounded compared to flat surfaces and associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell viability and proliferation was unchanged on nanorods. Adherent cell numbers were significantly decreased while single tumor cell motility was increased on nanorods compared to flat surfaces. Together, these results suggest that nanorods can be used to weaken malignant tumor cell adhesion, and therefore potentially improve tumor stent performance.

Advances in Hydrogen, Carbon Dioxide, and Hydrocarbon Gas Sensor Technology Using GaN and ZnO-Based Devices.

In this paper, we review our recent results in developing gas sensors for hydrogen using various device structures, including ZnO nanowires and GaN High Electron Mobility Transistors (HEMTs). ZnO nanowires are particularly interesting because they have a large surface area to volume ratio, which will improve sensitivity, and because they operate at low current levels, will have low power requirements in a sensor module. GaN-based devices offer the advantage of the HEMT structure, high temperature operation, and simple integration with existing fabrication technology and sensing systems. Improvements in sensitivity, recoverability, and reliability are presented. Also reported are demonstrations of detection of other gases, including CO(2) and C(2)H(4) using functionalized GaN HEMTs. This is critical for the development of lab-on-a-chip type systems and can provide a significant advance towards a market-ready sensor application.

GaN, ZnO and InN nanowires and devices.

A brief review is given of recent developments in wide bandgap semiconductor nanowire synthesis and devices fabricated on these nanostructures. There is strong interest in these devices for applications in UV detection, gas sensors and transparent electronics.

Targeted protein quantitation and profiling using PVDF affinity probe and MALDI-TOF MS.

Development of a rapid, effective, and highly specific platform for target identification in complex biofluids is one of the most important tasks in proteomic research. Taking advantage of the natural hydrophobic interaction of PVDF with probe protein, a simple and effective method was developed for protein quantitation and profiling. Using antibody-antigen interactions as a proof-of-concept system, the targeted plasma proteins, serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP), could be selectively isolated and enriched from human plasma by antibody-immobilized PVDF membrane and directly identified by MALDI-TOF MS without additional elution step. The approach was successfully applied to human plasma for rapid quantitation and variant screening of SAP, SAA, and CRP in healthy individuals and patients with gastric cancer. The triplexed on-probe quantitative analysis revealed significant overexpression of CRP and SAA in gastric cancer group, consistent with parallel ELISA measurements and pathological progression and prognostic significance reported in previous literatures. Furthermore, the variant mass profiling of the post-translationally modified forms revealed a high occurrence of de-sialic acid SAP in patients with gastric cancer. Due to the versatile assay design, ease of probe preparation without chemical synthesis, and compatibility with MALDI-TOF MS analysis, the methodology may be useful for target protein characterization, functional proteomics, and screening in clinical proteomics.

Androgenic and antiandrogenic effects and expression of androgen receptor in mouse embryonic stem cells.

OBJECTIVE: To investigate the effects of androgen and antiandrogen and the expression of androgen receptor on mouse embryonic stem cells (ESCs) and the inner cell mass. DESIGN: Controlled laboratory study. SETTING: Academic university hospital. ANIMAL(S): Blastocysts from mice developed at the Institute for Cancer Research and 129/Sv mice embryonic stem cell line. INTERVENTION(S): Cultured mouse ESCs were exposed to testosterone (T), dihydrotestosterone (DHT), or the antiandrogen nilutamide. MAIN OUTCOME MEASURE(S): Immunohistochemistry for androgen receptor (AR), quantitative real-time polymerase chain reaction analysis, cell colorimetric assays, and Western blot analysis. RESULT(S): Androgen receptor messenger RNA (mRNA) was first detected both in the inner cell mass from blastocysts and in undifferentiated ESCs. It increased stage-dependently during ESC differentiation. Although both T and DHT had marginal effects on AR mRNA expression level and cell growth in vitro, the nonsteroidal antiandrogen nilutamide significantly stimulated ESC growth and induced Akt expression. The enhancing effects of nilutamide on mouse ESCs indicated that the Akt pathway may be involved in nilutamide-promoted ESC growth. CONCLUSION(S): These findings provide the first evidence of the existence of AR in ESCs. During differentiation, the expression level of AR was increased in a stage-dependent but not a ligand-dependent manner. Nilutamide promoted cell growth and increased Akt expression in ESCs.

Improved analysis of membrane protein by PVDF-aided, matrix-assisted laser desorption/ionization mass spectrometry.

Characterization of membrane proteins remains an analytical challenge because of difficulties associated with tedious isolation and purification. This study presents the utility of the polyvinylidene difluoride (PVDF) membrane for direct sub-proteome profiling and membrane protein characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The hydrophobic adsorption of protein, particularly membrane proteins, on the PVDF surface enables efficient on-PVDF washing to remove high concentrations of detergents and salts, such as up to 5% sodium dodecyl sulfate (SDS). The enhanced spectrum quality for MALDI detection is particularly notable for high molecular weight proteins. By using on-PVDF washing prior to MALDI detection, we obtained protein profiles of the detergent-containing and detergent-insoluble membrane fractions from Methylococcus capsulatus (Bath). Similar improvements of signal-to-noise ratios were shown on the MALDI spectra for proteins electroblotted from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) onto the PVDF membrane. We have applied this strategy to obtain intact molecular weights of the particulate methane monooxygenase (pMMO) composed of three intrinsic membrane-bound proteins, PmoA, PmoB, and PmoC. Together with peptide sequencing by tandem mass spectrometry, post-translational modifications including N-terminal acetylation of PmoA and PmoC and alternative C-terminal truncation of PmoB were identified. The above results show that PVDF-aided MALDI-MS can be an effective approach for profiling and characterization of membrane proteins.

The influences of weather on patients with different ovarian responses in the treatment of assisted reproductive technology.

PURPOSE: The purpose of this study was to evaluate the influences of tropical weather on patients with different ovarian responses in the treatment of assisted reproductive technology. METHODS: Six-hundred fourty-seven women underwent their first treatment cycles were retrospectively analyzed. Patients received embryo transfer either 3 days or 5 days after oocyte retrieval, depending on the number and quality of embryos on day-2. RESULTS: Significant correlations were demonstrated in the top quality embryo rates of day-3 and day-5 embryo transfers with temperature, humidity, and atmosphere pressure. The cumulative light hours negatively correlated with the implantation and pregnancy rates of day-3 embryo transfer (-.282 and -.282, respectively), while they positively correlated with those of day-5 embryo transfer (.225 and .224, respectively). CONCLUSIONS: These results clearly suggest that weather may exert influences on the outcome of assisted reproductive technology. Patients with different ovarian responses or blastocyst culture and transfer may modify weather influences.