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1.  Evaluation of Postoperative Range of Motion and Functional Outcomes after Cruciate-Retaining and Posterior-Stabilized High-Flexion Total Knee Arthroplasty 
Yonsei Medical Journal  2012;53(4):794-800.
The purpose of this study was to compare postoperative range of motion and functional outcomes among patients who received high-flexion total knee arthroplasty using cruciate-retaining (CR-Flex) and posterior-stabilized (PS-Flex) type prostheses.
Materials and Methods
Among 127 patients (186 knees) who underwent high-flexion total knee arthroplasty between 2005 and 2007, 92 knees were placed in the CR-Flex group, and 94 knees were placed in the PS-Flex group. After two years of postoperative follow-up, clinical and radiographic data were reviewed. Postoperative non-weight-bearing range of knee motion, angle of flexion contracture and functional outcomes based on the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) functional sub-scale were assessed and compared between the two groups.
After the 2-year postoperative period, the mean range of motion was 131° in the CR-Flex group and 133° in the PS-Flex group. There were no significant differences in postoperative range of motion between the two groups. Only age at operation and preoperative range of motion were significantly associated with postoperative range of motion after high-flexion total knee arthroplasty. Postoperative functional outcomes based on the WOMAC functional sub-scale were slightly better in the CR-Flex group (9.2±9.1 points) than in the PS-Flex group (11.9±9.6 points); however, this difference was not statistically significant (p=non-significant).
The retention or substitution of the posterior cruciate ligament does not affect postoperative range of motion (ROM) or functional outcomes, according to 2 years of postoperative follow-up of high-flexion total knee arthroplasty.
PMCID: PMC3381472  PMID: 22665348
Total knee arthroplasty; range of motion; functional outcomes; cruciate-retaining; posterior-stabilized; high-flexion
2.  Long-Term Follow-Up Results of a Second-Generation Cementless Femoral Prosthesis with a Collar and Straight Distal Fixation Channels 
Yonsei Medical Journal  2011;53(1):186-192.
We evaluated the results of more than 10 years of follow-up of total hip arthroplasty using a second-generation cementless femoral prosthesis with a collar and straight distal fixation channels.
Materials and Methods
One hundred five patients (129 hips) who underwent surgery between 1991 and 1996 for primary total hip arthroplasty using cementless straight distal fluted femoral stems were followed for more than 10 years. Ninety-four hips in 80 patients were available for clinical and radiologic analysis. The mean age at the time of surgery was 47 years, and the mean duration of follow-up was 14.3 years.
The mean Harris hip scores had improved from 58 points to 88 points at the time of the 10-year follow-up. Activity-related thigh pain was reported in nine hips (10%). At the last follow-up, 93 stems (99%) were biologically stable and one stem (1%) was revised because of loosening. No hip had distal diaphyseal osteolysis. Proximal femoral stress-shielding was reported in 86 hips (91%). We found no significant relationship between collar-calcar contact and thigh pain, stem fixation status, or stress-shielding. The cumulative survival of the femoral stem was 99% (95% confidence interval, 98-100%) after 10 years.
The long-term results of total hip arthroplasty using a second-generation cementless femoral prosthesis with a collar and straight distal fixation channels were satisfactory; however, the high rate of proximal stress-shielding and the minimal effect of the collar indicate the need for some changes in the stem design.
PMCID: PMC3250326  PMID: 22187251
Total hip arthroplasty; second-generation; cementless; collar; straight distal fixation channel
3.  miR-150 Deficiency Protects against FAS-Induced Acute Liver Injury in Mice through Regulation of AKT 
PLoS ONE  2015;10(7):e0132734.
Although miR-150 is implicated in the regulation of immune cell differentiation and activation, it remains unknown whether miR-150 is involved in liver biology and disease. This study was performed to explore the potential role of miR-150 in LPS/D-GalN and Fas-induced liver injuries by using wild type and miR-150 knockout (KO) mice. Whereas knockout of miR-150 did not significantly alter LPS/D-GalN-induced animal death and liver injury, it protected against Fas-induced liver injury and mortality. The Jo2-induced increase in serum transaminases, apoptotic hepatocytes, PARP cleavage, as well as caspase-3/7, caspase-8, and caspase-9 activities were significantly attenuated in miR-150 KO mice. The liver tissues from Jo2-treated miR-150 KO mice expressed higher levels of Akt1, Akt2, total Akt, as well as p-Akt(Ser473) compared to the wild type livers. Pretreatment with the Akt inhibitor V reversed Jo2-induced liver injury in miR-150 KO mice. The primary hepatocytes isolated from miR-150 KO mice also showed protection against Fas-induced apoptosis in vitro (characterized by less prominent PARP cleavage, less nuclear fragmentation and less caspase activation) in comparison to hepatocytes from wild type mice. Luciferase reporter assays in hepatocytes transfected with the Akt1 or Akt2 3’-UTR reporter constructs (with or without mutation of miR-150 binding site) established Akt1 and Akt2 as direct targets of miR-150. Tail vein injection of lentiviral particles containing pre-miR-150 enhanced Jo2-induced liver injury in miR-150 KO mice. These findings demonstrate that miR-150 deficiency prevents Fas-induced hepatocyte apoptosis and liver injury through regulation of the Akt pathway.
PMCID: PMC4510058  PMID: 26196694
4.  Introgression of a 4D chromosomal fragment into durum wheat confers aluminium tolerance 
Annals of Botany  2014;114(1):135-144.
Background and Aim
Aluminium (Al3+) inhibits root growth of sensitive plant species and is a key factor that limits durum wheat (Triticum turgidum) production on acid soils. The aim of this study was to enhance the Al3+ tolerance of an elite durum cultivar by introgression of a chromosomal fragment from hexaploid wheat (Triticum aestivum) that possesses an Al3+ tolerance gene.
A 4D(4B) substitution line of durum wheat ‘Langdon’ was backcrossed to ‘Jandaroi’, a current semi-dwarf Australian durum. In the second backcross, using ‘Jandaroi’ as the recurrent parent, a seedling was identified where TaALMT1 on chromosome 4D was recombined with the Rht-B1b locus on chromosome 4B to yield an Al3+-tolerant seedling with a semi-dwarf habit. This seedling was used in a third backcross to generate homozygous sister lines with contrasting Al3+ tolerances. The backcrossed lines were characterized and compared with selected cultivars of hexaploid wheat for their Al3+ and Na+ tolerances in hydroponic culture as well as in short-term experiments to assess their growth on acid soil.
Key Results
Analysis of sister lines derived from the third backcross showed that the 4D chromosomal fragment substantially enhanced Al3+ tolerance. The ability to exclude Na+ from leaves was also enhanced, indicating that the chromosomal fragment possessed the Kna1 salt tolerance locus. Although Al3+ tolerance of seminal roots was enhanced in acid soil, the development of fine roots was not as robust as found in Al3+-tolerant lines of hexaploid wheat. Analysis of plant characteristics in the absence of Al3+ toxicity showed that the introgressed fragment did not affect total grain yield but reduced the weight of individual grains.
The results show that it is possible to increase substantially the Al3+ tolerance of an elite durum wheat cultivar by introgression of a 4D chromosomal fragment. Further improvements are possible, such as introducing additional genes to enhance the Al3+ tolerance of fine roots and by eliminating the locus on the chromosomal fragment responsible for smaller grain weights.
PMCID: PMC4071094  PMID: 24737716
Triticum turgidum; Triticum aestivum; durum wheat; 4D chromosomal fragment; aluminium tolerance; acid soil; root growth; salt tolerance; TaALMT1; Kna1; malate; rhizosheath
5.  Bathing Effects of Various Seawaters on Allergic (Atopic) Dermatitis-Like Skin Lesions Induced by 2,4-Dinitrochlorobenzene in Hairless Mice 
We evaluated the preventive effects of four types of seawater collected in Republic of Korea on hairless mice with 2,4-dinitrochlorobenzene- (DNCB-) induced allergic/atopic dermatitis (AD). The anti-inflammatory effects were evaluated by measuring tumor necrosis factor- (TNF-) α and interleukins (ILs). Glutathione (GSH), malondialdehyde (MDA), superoxide anion, and inducible nitric oxide synthase (iNOS) were measured to evaluate the antioxidant effects. Caspase-3 and poly (ADP-ribose) polymerase (PARP) were observed to measure the antiapoptotic effects; matrix metalloproteinase- (MMP-) 9 levels were also evaluated. Mice with AD had markedly higher clinical skin severity scores and scratching behaviors; higher TNF-α and ILs (1β, 10, 4, 5, and 13) levels; higher MDA, superoxide anion, caspase-3, PARP, and MMP-9 levels; and greater iNOS activity. However, the severity of AD was significantly decreased by bathing in seawaters, but it did not influence the dermal collagen depositions and skin tissue antioxidant defense systems. These results suggest that bathing in all four seawaters has protective effects against DNCB-induced AD through their favorable systemic and local immunomodulatory effects, active cytoprotective antiapoptotic effects, inhibitory effects of MMP activity and anti-inflammatory and antioxidative effects.
PMCID: PMC4488017  PMID: 26221169
6.  MiR-21 Targets 15-PGDH and Promotes Cholangiocarcinoma Growth 
Molecular cancer research : MCR  2014;12(6):890-900.
MicroRNAs (miRs) are a group of small, non-coding RNAs that modulate the translation of genes by binding to specific target sites in the target mRNA. This study investigated the biological function and molecular mechanism of microRNA-21 (miR-21) in human cholangiocarcinoma. In situ hybridization analysis of human cholangiocarcinoma specimens showed increased miR-21 in cholangiocarcinoma tissue compared to the non-cancerous biliary epithelium. Lentiviral transduction of miR-21 enhanced human cholangiocarcinoma cell growth and clonogenic efficiency in vitro, whereas inhibition of miR-21 decreased these parameters. Over-expression of miR-21 also promoted cholangiocarcinoma growth using an in vivo xenograft model system. The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (15-PGDH/HPGD), a key enzyme that converts the protumorigenic prostaglandin E2 (PGE2) to its biologically inactive metabolite, was identified as a direct target of miR-21 in cholangiocarcinoma cells. In parallel, cyclooxygenase-2 (COX2) over-expression and PGE2 treatment increased miR-21 levels and enhanced miR-21 promoter activity in human cholangiocarcinoma cells.
PMCID: PMC4058387  PMID: 24699315
Cholangiocarcinoma; miR-21; 15-PGDH; COX-2; PGE2
7.  Ulnar impaction syndrome with different operative methods: a comparative biomechanical study 
Objective: Ulnar impaction syndrome seriously impairs wrist and hand function. Three main treatment procedures are available; however, little systematic research on the post-operation changes in wrist biomechanics currently exists. This study aimed to determine the long-term effects of these procedures and the optimal treatment methods for ulnar impaction syndrome. Methods: Twenty-four cases of fresh upper limb specimens were randomized into four groups: (1) the control group, (2) the ulnar-shortening operation group, (3) the Sauvé-Kapandji procedure group (distal radioulnar arthrodesis and intentional distal ulnar pseudoarthrosis), and (4) the Darrach procedure group (distal ulna resection). After keeping the wrist in a neutral position, a pressure sensitive film was applied. Starting at 0 N, the load was increased gradually at a speed of 0.1 N/s until reaching 200 N and then maintained for 60 s by the CSS-44020 series biomechanical machine. Then, the pressure sensitive films from each group were measured, and the results were analyzed with SPSS software. Results: The mean pressure and force on the ulna in the groups followed a decreasing trend from the control group, Sauvé-Kapandji procedure group and ulnar-shortening operation group. The mean pressure of the scaphoid fossa and the force on distal aspect of the radius in the groups followed an increasing trend from the control group, Sauvé-Kapandji procedure group, ulnar-shortening operation group and Darrach procedure group. This study found no significant differences in the mean pressure of the scaphoid fossa and the force on distal aspect of the radius between the Sauvé-Kapandji procedure group and the ulnar-shortening operation group. The Sauvé-Kapandji procedure group showed the greatest mean pressure on lunate fossa. Conclusions: In this comprehensive analysis of wrist biomechanics, the ulnar-shortening operation was superior to the Sauvé-Kapandji procedure and Darrach procedure, which adequately maintained the anatomical relationships of the wrist.
PMCID: PMC4483933  PMID: 26131156
Biomechanics; the Darrach procedure; the Sauvé-Kapandji procedure; the ulnar-shortening operation; ulnar impaction syndrome
8.  Effects of Cholesterol Incorporation on the Physicochemical, Colloidal, and Biological Characteristics of pH-sensitive AB2 Miktoarm Polymer-Based Polymersomes 
In our previous study, a histidine-based AB2 miktoarm polymer, methoxy poly(ethylene glycol)-b-poly(l-histidine)2 (mPEG-b-(PolyHis)2), was designed to construct pH-sensitive polymersomes that transform in acidic pH; the polymer self-assembles into a structure that mimics phospholipids. In this study, the polymersomes further imitated liposomes due to the incorporation of cholesterol (CL). The hydrodynamic radii of the polymersomes increased with increasing CL wt% (e.g., 70 nm for 0 wt% vs. 91 nm for 1 wt%), resulting in an increased capacity for encapsulating hydrophilic drugs (e.g., 0.92 µL/mg for 0 wt% vs. 1.42 µL/mg for 1 wt%). The CL incorporation enhanced the colloidal stability of the polymersomes in the presence of serum protein and retarded their payload release. However, CL-incorporating polymersomes still demonstrated accelerated release of a hydrophilic dye (e.g., 5(6)-carboxyfluorescein (CF)) below pH 6.8 without losing their desirable pH sensitivity. CF-loaded CL-incorporating polymersomes showed better cellular internalization than the hydrophilic CF, whereas doxorubicin (DOX)-loaded CL-incorporating polymersomes presented similar or somewhat lower anti-tumor effects than free hydrophobic DOX. The findings suggest that CL-incorporating mPEG-b-(PolyHis)2-based polymersomes may have potential for intracellular drug delivery of chemical drugs due to their improved colloidal stability, lower drug loss during circulation, acidic pH-induced drug release, and endosomal disruption.
PMCID: PMC3995872  PMID: 24463148
Cholesterol; Colloidal stability; Miktoarm polymer; pH-sensitive; Poly(histidine); Polymersome
9.  Single Oral Dose Toxicity Test of Blue Honeysuckle Concentrate in Mice 
Toxicological Research  2015;31(1):61-68.
The objective of this study was to obtain single oral dose toxicity information for concentrated and lyophilized powder of blue honeysuckle (Lonicera caerulea L., Caprifoliaceae; BHcL) in female and male ICR mice to aid in the process of developing natural origin medicinal ingredients or foods following proximate analysis and phytochemical profile measurement. The proximate analysis revealed that BHcL had an energy value of 3.80 kcal/g and contained 0.93 g/g of carbohydrate, 0.41 g/g of sugar, 0.02 g/g of protein, and 0.20 mg/g of sodium. BHcL did not contain lipids, including saturated lipids, trans fats, or cholesterols. Further, BHcL contained 4.54% of betaine, 210.63 mg/g of total phenols, 159.30 mg/g of total flavonoids, and 133.57 mg/g of total anthocyanins. Following administration of a single oral BHcL treatment, there were no treatment-related mortalities, changes in body weight (bw) or organ weight, clinical signs, necropsy or histopathological findings up to 2,000 mg/kg bw, the limited dosage for rodents of both sexes. We concluded that BHcL is a practically non-toxic material in toxicity potency.
PMCID: PMC4395656  PMID: 25874034
Single oral dose toxicity; Mouse; Blue honeysuckle; Proximate analysis; Phytochemical analysis
10.  Dried Pomegranate Potentiates Anti-Osteoporotic and Anti-Obesity Activities of Red Clover Dry Extracts in Ovariectomized Rats 
Nutrients  2015;7(4):2622-2647.
Red clover (RC) shows potential activity against menopausal symptoms and pomegranates have antioxidative and beneficial effects on postmenopausal symptoms; thus, we investigated whether the anti-climacteric activity of RC could be enhanced by the addition of dried pomegranate concentrate powder (PCP) extracts in ovariectomized (OVX) rats. Regarding the anti-osteoporotic effects, bone mineral density increased significantly in OVX induced rats treated with 60 and 120 mg/kg of an RC:PCP 2:1 mixture, respectively, compared with OVX control rats. Additionally, femoral, tibia, and L4 bone resorption was decreased in OVX induced control rats treated with the RC:PCP 2:1 mixture (60 and 120 mg/kg), respectively, compared with OVX control rats. Regarding anti-obesity effects, the OVX induced rats treated with 60 and 120 mg/kg of the RC:PCP 2:1 mixture showed a decrease in total fat pad thickness, the mean diameters of adipocytes and the body weights gain compared with OVX induced control rats. The estradiol and bone-specific alkaline phosphatase levels were significantly increased in OVX induced rats treated with the RC:PCP 2:1 mixture (120 mg/kg) compared with OVX induced control rats, also, the uterine atrophy was significantly inhibited in 60 and 120 mg/kg of the RC:PCP 2:1 mixture treatment compared with OVX control rats. In conclusion, our results indicate that PCP enhanced the anti-climacteric effects of RC in OVX rats. The RC:PCP 2:1 mixture used in this study may be a promising new potent and protective agent for relieving climacteric symptoms.
PMCID: PMC4425164  PMID: 25912038
red clover extracts; dried pomegranate concentrate powder; 2:1 mixture; anti-climacteric effects; ovariectomy; rats
11.  Portable and Reusable Optofluidics-Based Biosensing Platform for Ultrasensitive Detection of Sulfadimidine in Dairy Products 
Sensors (Basel, Switzerland)  2015;15(4):8302-8313.
Sulfadimidine (SM2) is a highly toxic and ubiquitous pollutant which requires rapid, sensitive and portable detection method for environmental and food monitoring. Herein, the use for the detection of SM2 of a portable optofluidics-based biosensing platform, which was used for the accurate detection of bisphenol A, atrazine and melamine, is reported for the first time. The proposed compact biosensing system combines the advantages of an evanescent wave immunosensor and microfluidic technology. Through the indirect competitive immunoassay, the detection limit of the proposed optofluidics-based biosensing platform for SM2 reaches 0.05 μg·L−1 at the concentration of Cy5.5-labeled antibody of 0.1 μg·mL−1. Linearity is obtained over a dynamic range from 0.17 μg·L−1 to 10.73 μg·L−1. The surface of the fiber probe can be regenerated more than 300 times by means of 0.5% sodium dodecyl sulfate solution (pH = 1.9) washes without losing sensitivity. This method, featuring high sensitivity, portability and acceptable reproducibility shows potential in the detection of SM2 in real milk and other dairy products.
PMCID: PMC4431307  PMID: 25860072
optofluidics-based biosensing platform; optic fiber; sulfadimidine; dairy products
12.  MiR-122 in hepatitis B virus and hepatitis C virus dual infection 
World Journal of Hepatology  2015;7(3):498-506.
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the most common causes of chronic liver diseases and hepatocelluar carcinomas. Over the past few years, the liver-enriched microRNA-122 (miR-122) has been shown to differentially regulate viral replication of HBV and HCV. It is notable that the level of miR-122 is positively and negatively regulated by HCV and HBV, respectively. Consistent with the well-documented phenomenon that miR-122 promotes HCV accumulation, inhibition of miR-122 has been shown as an effective therapy for the treatment of HCV infection in both chimpanzees and humans. On the other hand, miR-122 is also known to block HBV replication, and HBV has recently been shown to inhibit miR-122 expression; such a reciprocal inhibition between miR-122 and HBV suggests an intriguing possibility that miR-122 replacement may represent a potential therapy for treatment of HBV infection. As HBV and HCV have shared transmission routes, dual infection is not an uncommon scenario, which is associated with more advanced liver disease than either HBV or HCV mono-infection. Thus, there is a clear need to further understand the interaction between HBV and HCV and to delineate the role of miR-122 in HBV/HCV dual infection in order to devise effective therapy. This review summarizes the current understanding of HBV/HCV dual infection, focusing on the pathobiological role and therapeutic potential of miR-122.
PMCID: PMC4381172  PMID: 25848473
MiR-122; Hepatitis B virus; Hepatitis C virus; Hepatitis B virus/hepatitis C virus dual infection
13.  Bright, stable, and water-soluble CuInS2/ZnS nanocrystals passivated by cetyltrimethylammonium bromide 
We report a highly bright and stable aqueous dispersion of CuInS2/ZnS (CIS/ZnS) nanocrystals (NCs) using surfactant-assisted microemulsion and cold treatment. CIS/ZnS NCs were facilely synthesized via a stepwise, consecutive hybrid flow reactor approach. To stabilize the optical properties of hydrophobic CIS/ZnS NCs, cetyltrimethylammonium bromide (CTAB) was chosen as a matrix for aqueous phase transfer. As the result, a high quantum yield (QY) of 56.0% and excellent photostability were acquired in aqueous media. For removing excessive surfactants, cold treatment (4°C) of the CTAB-water solution was adopted to prevent further agglomeration of CIS/ZnS NCs, which could secure high stability over 6 months (less 2% reduction in QY). The optical features and structure of the obtained CTAB stabilized CIS/ZnS (CTAB-CIS/ZnS) NCs have been characterized by UV–vis and photoluminescence (PL) spectroscopies, XRD, XPS, EDX, and TEM. The high stability and PL of water soluble CTAB-CIS/ZnS NCs suggest their potential in nanoelectronics and bioapplications.
PMCID: PMC4385304  PMID: 25852434
CuInS2/ZnS nanocrystals; CTAB; Photostability; Cold treatment
14.  Bone Marrow-Derived, Neural-Like Cells Have the Characteristics of Neurons to Protect the Peripheral Nerve in Microenvironment 
Stem Cells International  2015;2015:941625.
Effective repair of peripheral nerve defects is difficult because of the slow growth of new axonal growth. We propose that “neural-like cells” may be useful for the protection of peripheral nerve destructions. Such cells should prolong the time for the disintegration of spinal nerves, reduce lesions, and improve recovery. But the mechanism of neural-like cells in the peripheral nerve is still unclear. In this study, bone marrow-derived neural-like cells were used as seed cells. The cells were injected into the distal end of severed rabbit peripheral nerves that were no longer integrated with the central nervous system. Electromyography (EMG), immunohistochemistry, and transmission electron microscopy (TEM) were employed to analyze the development of the cells in the peripheral nerve environment. The CMAP amplitude appeared during the 5th week following surgery, at which time morphological characteristics of myelinated nerve fiber formation were observed. Bone marrow-derived neural-like cells could protect the disintegration and destruction of the injured peripheral nerve.
PMCID: PMC4378708  PMID: 25861281
15.  Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay 
Scientific Reports  2015;5:8125.
This work was the first to report that the kanamycin-binding DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA-3′) can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.
PMCID: PMC4311242  PMID: 25634469
16.  Epigenetic regulation of miR-122 by PPARγ and hepatitis B virus X protein in hepatocellular carcinoma cells 
Hepatology (Baltimore, Md.)  2013;58(5):1681-1692.
MiR-122, a pivotal liver specific miRNA, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR-122 in human hepatocellular carcinoma (HCC) cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR-122 as the most up-regulated miRNA (6-fold) in human hepatocellular cancer cells treated with 5′aza-2′deoxycytidine (5-Aza-CdR, DNA methylation inhibitor) and 4-phenylbutyric acid (PBA, histone deacetylation inhibitor). Real-time PCR analysis verified significant upregulation of miR-122 by 5′aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor-gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR-122 gene promoter which enhanced miR-122 gene transcription. 5-Aza-CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its co-repressors (N-CoR and SMRT), with the miR-122 DR1 and DR2 motifs. The aforementioned DNA-protein complex also contains SUV39H1, a H3K9 histone methyl transferase, which downregulates miR-122 expression. Our findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR-122 in human HCC cells. Moreover, we show that hepatitis B virus X protein (HBX) binds PPARγ and inhibits the transcription of miR-122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR-122 in patients with HBV but not with HCV infection.
PMCID: PMC3773012  PMID: 23703729
miR-122; PPARγ; HCC; epigenetic regulation; Hepatitis B virus X protein; HCV; liver; hepatocytes
17.  Suppression of lung inflammation in an LPS-induced acute lung injury model by the fruit hull of Gleditsia sinensis 
The fruit hull of Gleditsia sinensis (FGS) used in traditional Asian medicine was reported to have a preventive effect on lung inflammation in an acute lung injury (ALI) mouse model. Here, we explored FGS as a possible therapeutics against inflammatory lung diseases including ALI, and examined an underlying mechanism for the effect of FGS.
The decoction of FGS in water was prepared and fingerprinted. Mice received an intra-tracheal (i.t.) FGS 2 h after an intra-peritoneal (i.p.) injection of lipopolysaccharide (LPS). The effect of FGS on lung inflammation was determined by chest imaging of NF-κB reporter mice, counting inflammatory cells in bronchoalveolar lavage fluid, analyzing lung histology, and performing semi-quantitative RT-PCR analysis of lung tissue. Impact of Nrf2 on FGS effect was assessed by comparing Nrf2 knockout (KO) and wild type (WT) mice that were treated similarly.
Bioluminescence from the chest of the reporter mice was progressively increased to a peak at 16 h after an i.p. LPS treatment. FGS treatment 2 h after LPS reduced the bioluminescence and the expression of pro-inflammatory cytokine genes in the lung. While suppressing the infiltration of inflammatory cells to the lungs of WT mice, FGS post-treatment failed to reduce lung inflammation in Nrf2 KO mice. FGS activated Nrf2 and induced Nrf2-dependent gene expression in mouse lung.
FGS post-treatment suppressed lung inflammation in an LPS-induced ALI mouse model, which was mediated at least in part by Nrf2. Our results suggest a therapeutic potential of FGS on inflammatory lung diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/1472-6882-14-402) contains supplementary material, which is available to authorized users.
PMCID: PMC4203922  PMID: 25318387
Gleditsia sinensis; Traditional Asian medicine; Therapeutics; Acute lung inflammation; Nrf2
18.  NADPH Oxidase and the Cardiovascular Toxicity Associated with Smoking 
Toxicological Research  2014;30(3):149-157.
Smoking is one of the most serious but preventable causes of cardiovascular disease (CVD). Key aspects of pathological process associated with smoking include endothelial dysfunction, a prothrombotic state, inflammation, altered lipid metabolism, and hypoxia. Multiple molecular events are involved in smokinginduced CVD. However, the dysregulations of reactive oxygen species (ROS) generation and metabolism mainly contribute to the development of diverse CVDs, and NADPH oxidase (NOX) has been established as a source of ROS responsible for the pathogenesis of CVD. NOX activation and resultant ROS production by cigarette smoke (CS) treatment have been widely observed in isolated blood vessels and cultured vascular cells, including endothelial and smooth muscle cells. NOX-mediated oxidative stress has also been demonstrated in animal studies. Of the various NOX isoforms, NOX2 has been reported to mediate ROS generation by CS, but other isoforms were not tested thoroughly. Of the many CS constituents, nicotine, methyl vinyl ketone, and α,β-unsaturated aldehydes, such as, acrolein and crotonaldehyde, appear to be primarily responsible for NOX-mediated cytotoxicity, but additional validation will be needed. Human epidemiological studies have reported relationships between polymorphisms in the CYBA gene encoding p22phox, a catalytic subunit of NOX and susceptibility to smoking-related CVDs. In particular, G allele carriers of A640G and -930A/G polymorphisms were found to be vulnerable to smoking-induced cardiovascular toxicity, but results for C242T studies are conflicting. On the whole, evidence implicates the etiological role of NOX in smoking-induced CVD, but the clinical relevance of NOX activation by smoking and its contribution to CVD require further validation in human studies. A detailed understanding of the role of NOX would be helpful to assess the risk of smoking to human health, to define high-risk subgroups, and to develop strategies to prevent or treat smoking-induced CVD.
PMCID: PMC4206741  PMID: 25343008
Smoking; Cigarette; NADPH oxidase; Reactive oxygen species; Oxidative stress; Cardiovascular disease
19.  The Sensitivity of Cancer Cells to Pheophorbide a-Based Photodynamic Therapy Is Enhanced by NRF2 Silencing 
PLoS ONE  2014;9(9):e107158.
Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.
PMCID: PMC4165896  PMID: 25226504
20.  Hedgehog Signaling Pathway Regulates Autophagy in Human Hepatocellular Carcinoma Cells 
Hepatology (Baltimore, Md.)  2013;58(3):995-1010.
Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunobloting for microtubule-associated protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining and transmission electron microscopy. Hh inhibition-induced autophagy was associated with upregulation of Bnip3, as determined by immunoblotting and real-time PCR assay. Knockdown of Bnip3 by RNAi impaired GANT61-induced autophagy. Additionally, Hh inhibition-induced autophagy was associated with Bnip3-mediated displacement of Bcl-2 from Beclin-1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST-1 assays. Pharmacological or genetic inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 siRNA partially suppressed GANT61-induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3-MA. These findings provide novel evidence that hedgehog inhibition induces autophagy through upregulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh-targeted therapies.
PMCID: PMC3706478  PMID: 23504944
Hedgehog signaling; GANT61; autophagy; hepatocellular carcinoma; apoptosis
21.  Minimum 5-year Follow-up Results of Minimally Invasive Total Knee Arthroplasty Using Mini-Keel Modular Tibial Implant 
Knee Surgery & Related Research  2014;26(3):149-154.
To evaluate the minimum 5-year mid-term clinical and radiological results of minimally invasive surgery total knee arthroplasty (MIS-TKA) using a mini-keel modular tibia component.
Materials and Methods
We retrospectively evaluated 254 patients (361 cases) who underwent MIS-TKA between 2005 and 2006. The latest clinical and radiological assessments were done in 168 cases that had been followed on an outpatient basis for more than 5 postoperative years. Clinical results were assessed using the Hospital for Special Surgery (HSS) score and Knee Society score. Radiological evaluation included measurements of knee alignment.
The average postoperative knee range of motion and HSS score were 134.3°±12.4° and 92.7°±7.0°, respectively. The average postoperative femorotibial angle and tibial component alignment angle were 5.2°±1.7° valgus and 90.2°±1.6°, respectively. The average tibial component posterior inclination was 4.8°±2.1°. The percentage of cases with tibial component alignment angle of 90°±3° was 96.1%, and that with the femorotibial angle of 6°±3° valgus was 94.0%. Radiolucent lines were observed in 20 cases (12.0%): around the femur, tibia, and patella in 14 cases, 10 cases, and 1 case, respectively. However, they were less than 2 mm and non-progressive in all cases. The survival rate was 99.4% and there was no implant-related revision.
MIS-TKA using a mini-keel modular tibial plate showed satisfactory results, a high survival rate, and excellent clinical and radiological results in the mid-term follow-up.
PMCID: PMC4163572  PMID: 25229044
Knee; Arthroplasty; Minimally invasive; Mini-keel tibial component
22.  Fermentation with Aquilariae Lignum Enhances the Anti-Diabetic Activity of Green Tea in Type II Diabetic db/db Mouse 
Nutrients  2014;6(9):3536-3571.
The major components of tea may be significantly influenced according to the type of fermentation, and consequently the effects of different teas will differ. We examined whether green tea fermented with Aquilariae Lignum (fGT) shows a stronger anti-diabetic effect than unfermented green tea (GT) on mice with type 2 diabetes. To evaluate the anti-obesity effect of fGT, we assessed body weight, fecal excretion, serum leptin levels, exocrine pancreatic zymogen granule contents, and periovarian fat weight and adiponectin contents. Blood glucose levels, pancreatic weight, and numbers of pancreatic islet insulin- and glucagon-producing cells were determined to evaluate anti-hypoglycemic effects, while total cholesterol, triglyceride, and low- and high-density lipoprotein levels were determined to evaluate anti-hyperlipidemic effects. The antioxidant effect of fGT was detected by measuring malondialdehyde and glutathione contents and the activities of catalase and superoxide dismutase. fGT showed anti-obesity, anti-hypoglycemic, anti-hyperlipidemia, and antioxidant effects. Additionally, fGT exerted stronger anti-diabetic effects compared with GT. Collectively, these results suggested that fGT fermented with the appropriate amounts of Aquilariae Lignum (49:1) has a stronger effect compared with GT. Thus, fGT is a promising and potent new therapeutic agent for type 2 diabetes.
PMCID: PMC4179175  PMID: 25207824
BKS.Cg-+Leprdb/+Leprdb/OlaHsd (db/db) mice; obese; diabetes; fermented green tea with Aquilariae Lignum; synergistic
23.  15-PGDH inhibits hepatocellular carcinoma growth through 15-keto-PGE2/PPARγ-mediated activation of p21WAF1/Cip1 
Oncogene  2013;33(9):1101-1112.
15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in prostaglandin metabolism. This study provides important evidence for inhibition of hepatocellular carcinoma (HCC) growth by 15-PGDH through the 15-keto-PGE2/PPARγ/p21WAF1/Cip1 signaling pathway. Forced overexpression of 15-PGDH inhibited HCC cell growth in vitro, whereas knockdown of 15-PGDH enhanced tumor growth parameters. In a tumor xenograft model in SCID mice, inoculation of human HCC cells (Huh7) with overexpression of 15-PGDH led to significant inhibition of tumor growth, while knockdown of 15-PGDH enhanced tumor growth. In a separate tumor xenograft model in which mouse HCC cells (Hepa1-6) were inoculated into syngeneic C57BL/6 mice, intratumoral injection of adenovirus vector expressing 15-PGDH (pAd-15-PGDH) significantly inhibited xenograft tumor growth. The anti-tumor effect of 15-PGDH is mediated through its enzymatic product, 15-keto-PGE2, which serves as an endogenous PPARγ ligand. Activation of PPARγ by 15-PGDH-derived 15-keto-PGE2 enhanced the association of PPARγ with the p21WAF1/Cip1 promoter and increased p21 expression and association with CDK2, CDK4 and PCNA. Depletion of p21 by shRNA reversed 15-PGDH-induced inhibition of HCC cell growth; overexpression of p21 prevented 15-PGDH knockdown-induced tumor cell growth. These results demonstrate a key 15-PGDH/15-keto-PGE2-mediated activation of PPARγ and p21WAF1/Cip1 signaling cascade that regulates hepatocarcinogenesis and tumor progression.
PMCID: PMC3912217  PMID: 23542179
Hepatocellular carcinoma (HCC); 15-hydroxyprostaglandin dehydrogenase (15-PGDH); 15-keto-PGE2; p21; PPARγ; liver
24.  Endosomolytic reducible polymeric electrolytes for cytosolic protein delivery 
Biomacromolecules  2013;14(8):2570-2581.
Despite the numerous vital functions of proteins in the cytosolic compartment, less attention has been paid to the delivery of protein drugs to the cytosol than to the plasma membrane. To address this issue and effectively deliver charged proteins into the cytoplasm, we used endosomolytic, thiol-triggered degradable polyelectrolytes as carriers. The cationic, reducible polyelectrolyte RPC-bPEI0.8kDa2 was synthesized by the oxidative polymerization of thiolated branched polyethyleneimine (bPEI). The polymer was converted to the anionic, reducible polyelectrolyte RPA-bPEI0.8kDa2 by introducing carboxylic acids. The two reducible polyelectrolytes (RPC-bPEI0.8kDa2 and RPA-bPEI0.8kDa2) were complexed with counter-charged model proteins (bovine serum albumin (BSA) and lysozyme (LYZ)), forming polyelectrolyte/protein complexes of less than 200 nm in size at weight ratios (WR) of ≥ 1. The resultant complexes maintained a proton buffering capacity nearly equivalent to that of the polyelectrolytes in the absence of protein complexation and were cytocompatible with MCF7 human breast carcinoma cells. Under cytosol-mimicking thiol-rich conditions, RPC-bPEI0.8kDa2/BSA and RPA-bPEI0.8kDa2/LYZ complexes increased significantly in size and released the loaded protein, unlike the protein complexes with non-reducible polyelectrolytes (bPEI25kDa and bPEI25kDaCOOH). The polyelectrolyte/protein complexes showed similar cellular uptake to the corresponding proteins alone, but the former allowed more protein to escape into the cytosol from endolysosomes than the latter as a result of the endosomolytic function of the polyelectrolytes. In addition, the proteins in the polyelectrolyte/protein complexes kept their intrinsic secondary structures. In conclusion, the results show the potential of the designed endosomolytic, reducible polyelectrolytes for the delivery of proteins to the cytosol.
PMCID: PMC3759265  PMID: 23841591
Cytosolic delivery; Endosomolytic polymer; Polyelectrolyte; Polyethyleneimine; Protein delivery; Reducible polymer
25.  Isoniazid Could Be Used for Antibiotic-loaded Bone Cement for Musculoskeletal Tuberculosis: An In Vitro Study 
Antibiotic-loaded bone cement (ALBC) has been used in serious cases of musculoskeletal tuberculosis, but the type and amount of antibiotic that should be used in ALBC have not been determined.
We therefore determined the (1) elution characteristics and (2) antimycobacterial activity of isoniazid- and rifampicin-loaded bone cement.
A total of 240 elution samples of each of three discs from 40 g bone cement mixed with one of eight dosages: 1 g, 2 g, and 4 g isoniazid, 1 g, 2 g, and 4 g rifampicin, and a combination of 1 + 1 g or 2 + 2 g of isoniazid and rifampicin. The polymerization of rifampicin-loaded bone cement was delayed to mean 122.5 ± 31.1 minutes. We measured the quantity of isoniazid and rifampicin and the antimycobacterial activity on Days 1, 3, 7, 14, and 30.
Isoniazid eluted in almost all the samples while rifampicin was detected only on Day 1 with 2 g (0.7 ± 0.4 ug/mL/day), and until Day 14 with 4 g (0.1 ± 0.0u g/mL/day). Most of the samples containing isoniazid showed antimycobacterial activity while the samples containing rifampicin showed antimycobacterial activity only on Day 1 with 1 g (0.52 ± 0.18 ug/mL), until Day 14 with 2 g (0.03 ± 0.00 ug/mL), and until Day 30 with 4 g (1.84 ± 1.90 ug/mL).
Rifampicin was unsuitable for ALBC because of its delayed polymerization. Isoniazid eluted and showed antimycobacterial activity for 30 days.
Clinical Relevance
The data suggest isoniazid could be considered for use in ALBC for musculoskeletal tuberculosis if used with systemic treatment. For preventing resistance and systemic toxicity, a combination with a second-line drug and an in vivo study would be needed.
PMCID: PMC3676586  PMID: 23504536

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