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1.  Clinical Outcomes of CyberKnife Radiotherapy in Prostate Cancer Patients: Short-term, Single-Center Experience 
Korean Journal of Urology  2014;55(3):172-177.
In this retrospective study, we analyzed the outcomes of prostate cancer patients treated with the CyberKnife radiotherapy system (Accuray).
Materials and Methods
Between 2007 and 2010, 31 patients were treated for prostate cancer by use of the CyberKnife radiotherapy system. After excluding six patients who were lost to follow-up, data for the remaining 25 patients were analyzed. Patients were divided into the CyberKnife monotherapy group and a postexternal beam radiotherapy boost group. Clinicopathologic features and treatment outcomes were compared between the groups. The primary endpoint was biochemical recurrence-free survival period based on the Phoenix definition. Toxicities were evaluated by using the Radiation Therapy Oncology Group scoring criteria.
Of 25 patients, 17 (68%) and 8 (32%) were classified in the monotherapy and boost groups, respectively. With a median follow-up of 29.3 months, most of the toxicities were grade 1 or 2 except for one patient in the boost group who experienced late grade 3 gastrointestinal toxicity. The overall biochemical recurrence rate was 20% (5/25) and the median time to biochemical recurrence was 51.9 months. None of the patients with low or intermediate risk had experienced biochemical recurrence during follow-up. Among D'Amico high-risk populations, 16.7% (1/6) in the monotherapy group and 50.0% (4/8) in the boost group experienced biochemical recurrence.
Our data support that prostate cancer treatment by use of the CyberKnife radiotherapy system is feasible. The procedure can be a viable option for managing prostate cancer either in a monotherapy setting or as a boost after conventional radiotherapy regardless of the patient's risk stratification.
PMCID: PMC3956945  PMID: 24648871
Prostate cancer; Recurrence; Stereotactic radiosurgery; Toxicity
2.  Dermatomyositis associated with hepatitis B virus-related hepatocellular carcinoma 
Dermatomyositis is an idiopathic inflammatory myopathy with typical cutaneous manifestations. It has been proposed that dermatomyositis may be caused by autoimmune responses to viral infections. Previous studies have shown an association between dermatomyositis and malignant tumors such as ovarian cancer, lung cancer, and colorectal cancer. However, a chronic hepatitis B virus (HBV) infection associated with dermatomyositis and hepatocellular carcinoma (HCC) has been very rarely reported. Here, we report a rare case of dermatomyositis coinciding with HBV-associated HCC. A 55-year-old male was confirmed to have HCC and dermatomyositis based on proximal muscle weakness, typical skin manifestations, elevated muscle enzyme levels, and muscle biopsy findings. This case suggests that HCC and/or a chronic HBV infection may be factors in the pathogenesis of dermatomyositis through a paraneoplastic mechanism.
PMCID: PMC3956994  PMID: 24648807
Dermatomyositis; Hepatitis B virus; Hepatocellular carcinoma
3.  Gene Expression Profiles of Human Adipose Tissue-Derived Mesenchymal Stem Cells Are Modified by Cell Culture Density 
PLoS ONE  2014;9(1):e83363.
Previous studies conducted cell expansion ex vivo using low initial plating densities for optimal expansion and subsequent differentiation of mesenchymal stem cells (MSCs). However, MSC populations are heterogeneous and culture conditions can affect the characteristics of MSCs. In this study, differences in gene expression profiles of adipose tissue (AT)-derived MSCs were examined after harvesting cells cultured at different densities. AT-MSCs from three different donors were plated at a density of 200 or 5,000 cells/cm2. After 7 days in culture, detailed gene expression profiles were investigated using a DNA chip microarray, and subsequently validated using a reverse transcription polymerase chain reaction (RT-PCR) analysis. Gene expression profiles were influenced primarily by the level of cell confluence at harvest. In MSCs harvested at ∼90% confluence, 177 genes were up-regulated and 102 genes down-regulated relative to cells harvested at ∼50% confluence (P<0.05, FC>2). Proliferation-related genes were highly expressed in MSCs harvested at low density, while genes that were highly expressed in MSCs harvested at high density (∼90% confluent) were linked to immunity and defense, cell communication, signal transduction and cell motility. Several cytokine, chemokine and growth factor genes involved in immunosuppression, migration, and reconstitution of damaged tissues were up-regulated in MSCs harvested at high density compared with MSCs harvested at low density. These results imply that cell density at harvest is a critical factor for modulating the specific gene-expression patterns of heterogeneous MSCs.
PMCID: PMC3882209  PMID: 24400072
4.  Important predictor of mortality in patients with end-stage liver disease 
Clinical and molecular hepatology  2013;19(2):105-115.
Prognosis is an essential part of the baseline assessment of any disease. For predicting prognosis of end-stage liver disease, many prognostic models were proposed. Child-Pugh score has been the reference for assessing the prognosis of cirrhosis for about three decades in end-stage liver disease. Despite of several limitations, recent large systematic review showed that Child-Pugh score was still robust predictors and it's components (bilirubin, albumin and prothrombin time) were followed by Child-Pugh score. Recently, Model for end-stage liver disease (MELD) score emerged as a "modern" alternative to Child-Pugh score. The MELD score has been an important role to accurately predict the severity of liver disease and effectively assess the risk of mortality. Due to several weakness of MELD score, new modified MELD scores (MELD-Na, Delta MELD) have been developed and validated. This review summarizes the current knowledge about the prognostic factors in end-stage liver disease, focusing on the role of Child-Pugh and MELD score.
PMCID: PMC3701842  PMID: 23837134
Cirrhosis; MELD score; Child score
5.  Durability of viral response after off-treatment in HBeAg positive chronic hepatitis B 
AIM: To evaluate the durability in hepatitis B e antigen (HBeAg) positive chronic hepatitis B patients who discontinued antiviral treatment.
METHODS: A total of 48 HBeAg positive chronic hepatitis B patients who were administered nucleoside analogues and maintained virological response for ≥ 6 mo [hepatitis B virus (HBV) DNA < 300 copies/mL and HBeAg seroconversion] before cessation of treatment were enrolled between February 2007 and January 2010. The criteria for the cessation of the antiviral treatment were defined as follows: (1) achievement of virological response; and (2) duration of consolidation therapy (≥ 6 mo). After treatment cessation, the patients were followed up at 3-6 mo intervals. The primary endpoint was serologic and virologic recurrence rates after withdrawal of antiviral treatment. Serologic recurrence was defined as reappearance of HBeAg positivity after HBeAg seroconversion. Virologic recurrence was defined as an increase in HBV-DNA level > 104 copies/mL after HBeAg seroconversion with previously undetectable HBV-DNA level.
RESULTS: During the median follow-up period of 18.2 mo (range: 5.1-47.5 mo) after cessation of antiviral treatment, the cumulative serological recurrence rate was 15 % at 12 mo. The median duration between the cessation of antiviral treatment and serologic recurrence was 7.2 mo (range: 1.2-10.9 mo). Of the 48 patients with HBeAg positive chronic hepatitis, 20 (41.6%) showed virological recurrence. The cumulative virologic recurrence rates at 12 mo after discontinuing the antiviral agent were 41%. The median duration between off-treatment and virologic recurrence was 7.6 mo (range: 4.3-27.1 mo). The mean age of the virological recurrence group was older than that of the non-recurrence group (46.7 ± 12.1 years vs 38.8 ± 12.7 years, respectively; P = 0.022). Age (> 40 years) and the duration of consolidation treatment (≥ 15 mo) were significant predictive factors for offtreatment durability in the multivariate analysis [P = 0.049, relative risk (RR) 0.31, 95% CI (0.096-0.998) and P = 0.005, RR 11.29, 95% CI (2.054-65.12), respectively]. Patients with age (≤ 40 years) who received consolidation treatment (≥ 15 mo) significantly showed durability in HBeAg positive chronic hepatitis B patients (P = 0.014). These results suggest that additional treatment for more than 15 mo after HBeAg seroconversion in patients who are ≤ 40 years old may be beneficial in providing a sustained virological response.
CONCLUSION: Our data suggest that HBeAg seroconversion is an imperfect end point in antiviral treatment. Long-term consolidation treatment (≥ 15 mo) in younger patients is important for producing better prognosis in HBeAg positive chronic hepatitis B.
PMCID: PMC3501777  PMID: 23180949
Durability; Seroconversion; Chronic hepatitis B; Hepatitis B e antigen positive; Recurrence; Consolidation
6.  A case of hypereosinophilic syndrome presenting with intractable gastric ulcers 
We report a rare case of hypereosinophilic syndrome (HES) presenting with intractable gastric ulcers. A 71-year-old man was admitted with epigastric pain. Initial endoscopic findings revealed multiple, active gastric ulcers in the gastric antrum. He underwent Helicobacter pylori (H pylori) eradication therapy followed by proton pump inhibitor (PPI) therapy. However, follow-up endoscopy at 4, 6, 10 and 14 mo revealed persistent multiple gastric ulcers without significant improvement. The proportion of his eosinophil count increased to 43% (total count: 7903/mm3). Abdominal-pelvic and chest computed tomography scans showed multiple small nodules in the liver and both lungs. The endoscopic biopsy specimen taken from the gastric antrum revealed prominent eosinophilic infiltration, and the liver biopsy specimen also showed eosinophilic infiltration in the portal tract and sinusoid. A bone marrow biopsy disclosed eosinophilic hyperplasia as well as increased cellularity of 70%. The patient was finally diagnosed with HES involving the stomach, liver, lung, and bone marrow. When gastric ulcers do not improve despite H pylori eradication and prolonged PPI therapy, infiltrative gastric disorders such as HES should be considered.
PMCID: PMC2797674  PMID: 20027690
Gastric ulcer; Hypereosinophilic syndrome
7.  Long-term outcomes of two rescue therapies in lamivudine-refractory patients with chronic hepatitis B: combined lamivudine and adefovir, and 1-mg entecavir 
Clinical and Molecular Hepatology  2014;20(3):267-273.
Adefovir (ADV) and lamivudine (LAM) combination therapy (ADV+LAM) has been a useful option for patients with LAM-resistant (LAM-r) chronic hepatitis B (CHB). However, the long-term outcomes of LAM+ADV and 1-mg entecavir (ETV) rescue therapies have still been limited. The aim of this study was to determine the long-term outcomes of these two rescue therapies.
Sixty patients with LAM-r CHB underwent rescue therapy with LAM+ADV (n=36) or 1-mg ETV (n=24). We determined the duration of rescue therapy, timing and type of mutation, undetectable serum hepatitis B virus (HBV) DNA by PCR (lower limitation of detection, < 140 copies/mL), biochemical response (alanine aminotransferase < 40 IU/mL), and the incidence of hepatitis B virus e antigen (HBeAg) seroconversion and virologic breakthrough.
Baseline characteristics did not differ between the two therapy groups. The duration of rescue therapy was 56 months (range, 14-100 months) in the ADV+LAM group and 42 months (range, 12-73 months) in the ETV group (P=0.036). The cumulative rates of HBV DNA undetectability and HBeAg seroconversion up to 6 years were 88.6% and 43.0%, respectively, in the ADV+LAM group, and 45.8% and 31.8% in the ETV group. The rate of virologic breakthrough and resistance was 14.4% in the ADV+LAM group and 71.9% in the ETV group (P=0.001).
Combination of LAM and ADV therapy for up to 6 years achieved modest rates of virological suppression and resistance. ETV is not an optimal therapy because the risk of viral breakthrough to ETV increases over time.
PMCID: PMC4197175  PMID: 25320730
Chronic hepatitis B; Lamivudine; Adefovir; Entecavir; Resistance
8.  Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation 
Scientific Reports  2014;4:5923.
Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.
PMCID: PMC4118148  PMID: 25080919
9.  Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System 
Molecules and Cells  2014;37(6):497-502.
Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.
PMCID: PMC4086344  PMID: 24938227
axons; human embryonic stem cells; microfluidics; migration; neural differentiation
10.  The Impacts of C-Reactive Protein and Atrial Fibrillation on Carotid Atherosclerosis and Ischemic Stroke in Patients with Suspected Ischemic Cerebrovascular Disease: A Single-Center Retrospective Observational Cohort Study 
Korean Circulation Journal  2013;43(12):796-803.
Background and Objectives
Carotid intima-media thickness (IMT) is associated with chronic inflammation, and C-reactive protein (CRP) level is elevated in patients with atrial fibrillation (AF). We investigated the impacts of CRP and AF on carotid atherosclerosis and ischemic stroke in patients with suspected ischemic cerebrovascular disease.
Subjects and Methods
One-hundred forty patients (78 males) with suspected ischemic cerebrovascular disease underwent carotid ultrasonography. The mean common carotid artery IMT, mean internal carotid artery (ICA) IMT, and plaque score were measured. Patients were divided into four groups according to the presence of AF and elevated CRP level {n=46 for AF(-)CRP(-), n=38 for AF(-)CRP(+), n=43 for AF(+)CRP(-), and n=13 for AF(+)CRP(+)}.
Common carotid artery IMT was significantly higher in the AF(-)CRP(+) (0.98±0.51 mm) and AF(+)CRP(+) (0.96±0.27 mm) groups compared to the AF(-)CRP(-) (0.80±0.32 mm) and AF(+)CRP(-) (0.77±0.19 mm) groups (p=0.027). Although there was no significant difference in mean ICA IMT among the groups, plaque score was the highest in the AF(+)CRP(+) (4.18±3.84 mm) group, followed by AF(-)CRP(+) (3.87±2.78 mm), AF(+)CRP(-) (1.34±2.61 mm), and AF(-)CRP(-) (1.17±2.02 mm) (p<0.001). The AF(+)CRP(+) group showed significantly higher incidence of ischemic stroke than the other groups (all p<0.05). Binary logistic regression analysis showed that age {odds ratio (OR)=1.033, p=0.001}, elevated CRP (OR=3.884, p=0.001), and the presence of AF (OR=1.375, p=0.018) were significantly correlated with incidence of ischemic stroke.
Elevated plasma CRP concentration may be a reliable surrogate marker for predicting carotid atherosclerosis in patients with AF, which may be related to increased risk of ischemic stroke.
PMCID: PMC3875695  PMID: 24385990
Atrial fibrillation; C-reactive protein; Carotid atherosclerosis
11.  An Unusual Case of Superior Vena Cava Syndrome Caused by the Intravascular Invasion of an Invasive Thymoma 
Superior vena cava syndrome (SVCS) is usually caused by extrinsic compression or invasion of the superior vena cava (SVC) by malignant tumors involving mediastinal structures. Although thymomas are well-known causes of SVCS, cases of SVCS caused by malignant thymomas protruding into adjacent vessels draining the SVC with thrombosis have been very rarely reported worldwide. We experienced a 39-year-old female patient with SVCS that developed after the direct invasion of the left brachiocephalic vein (LBCV) and SVC by an anterior mediastinal mass with a high maximum standardized uptake value on the chest computed tomography (CT) and positron emission tomography-CT. Based on these results, she underwent en bloc resection of the tumor, including removal of the involved vessels, and was eventually diagnosed as having a type B2 thymoma permeating into the LBCV and SVC. We present this case as a very rare form of SVCS caused by an invasive thymoma.
PMCID: PMC3861377  PMID: 24348669
Thymoma; Superior Vena Cava Syndrome; Positron-Emission Tomography
12.  Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival 
The Journal of Cell Biology  2012;199(7):1145-1158.
Plasma membrane calcium ATPases PMCA1 and PMCA4 regulate osteoclast differentiation and survival by regulating NFATc1 and NO.
The precise regulation of Ca2+ dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca2+ ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand–induced Ca2+ oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca2+ efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca2+ signaling in osteoclasts.
PMCID: PMC3529522  PMID: 23266958
13.  Quantitative Analysis of Axonal Transport by Using Compartmentalized and Surface Micropatterned Culture of Neurons 
ACS Chemical Neuroscience  2012;3(6):433-438.
Mitochondria, synaptic vesicles, and other cytoplasmic constituents have to travel long distance along the axons from cell bodies to nerve terminals. Interruption of this axonal transport may contribute to many neurodegenerative diseases including Alzheimer’s disease (AD). It has been recently shown that exposure of cultured neurons to β-amyloid (Aβ) resulted in severe impairment of mitochondrial transport. This Letter describes an integrated microfluidic platform that establishes surface patterned and compartmentalized culture of neurons for studying the effect of Aβ on mitochondria trafficking in full length of axons. We have successfully quantified the trafficking of fluorescently labeled mitochondria in distal and proximal axons using image processing. Selective treatment of Aβ in the somal or axonal compartments resulted in considerable decrease in mitochondria movement in a location dependent manner such that mitochondria trafficking slowed down more significantly proximal to the location of Aβ exposure. Furthermore, this result suggests a promising application of microfluidic technology for investigating the dysfunction of axonal transport related to neurodegenerative diseases.
PMCID: PMC3643179  PMID: 24358503
β-Amyloid; axonal transport; mitochondrial trafficking; microfludics; surface micropatterning; image processing
14.  Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy 
Nature neuroscience  2012;15(12):1742-1751.
Protein synthesis is highly regulated throughout nervous system development, plasticity, and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.
PMCID: PMC3509268  PMID: 23103964
15.  Predictive Preoperative Factors for Renal Insufficiency in Patients Followed for More Than 5 Years After Radical Nephrectomy 
Korean Journal of Urology  2013;54(5):303-310.
We assessed the predictive factors for renal insufficiency in patients followed for more than 5 years after radical nephrectomy.
Materials and Methods
Age, gender, history of diabetes, history of hypertension, body mass index, preoperative estimated glomerular filtration rate (eGFR), serum uric acid, urine albumin, normal renal parenchymal volume, tumor size, and ratio of normal parenchymal volume of the removed kidney to that of the remaining kidney were evaluated retrospectively in 89 patients who underwent radical nephrectomy from January 2001 to December 2005. Patients were included whose renal parenchymal volume was measurable by use of perioperative imaging (computed tomography or magnetic resonance imaging), whose preoperative eGFR was greater than 60 mL/min/1.73 m2, and who were followed for more than 5 years. To measure renal parenchymal volume from imaging, we integrated the extent of the normal renal parenchyma from axial slides of images.
In univariate and multivariate binary regression analysis, the parenchymal volume of the remnant kidney (p=0.001), a history of diabetes (p=0.035), and preoperative eGFR (p=0.011) were independent factors for renal insufficiency. By use of a receiver operating characteristic curve, a volume of 170 mL was determined to be an appropriate cutoff value, with sensitivity of 58.7% and specificity of 74.4% for the parenchymal volume of the remnant kidney for predicting eGFR less than 60 mL/min/1.73 m2 (area under the curve, 0.678). The parenchymal volume of the remnant kidney was also an independent factor for the downgrading of the chronic kidney disease category in the multivariate linear regression analysis (p=0.021).
Preoperative eGFR, a history of diabetes, and the radiologic volume of the remaining kidney parenchyma could be useful factors for predicting postoperative renal function. Patients with parenchymal volumes of less than 170 mL have a higher risk of postoperative renal insufficiency, which should be considered carefully when choosing a treatment modality.
PMCID: PMC3659223  PMID: 23700495
Kidney; Nephrectomy; Organ size; Renal insufficiency
16.  Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction 
Molecular Biology of the Cell  2013;24(4):521-534.
G-proteins, heterotrimeric and Cdc42, modulate in a ligand-dependent fashion two fundamental cell polarity behaviors (projection bending growth and second projection formation) in response to gradient directional change.
Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change.
PMCID: PMC3571874  PMID: 23242998
17.  Reprogramming axonal behavior by axon-specific viral transduction 
Gene therapy  2012;19(9):947-955.
The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.
PMCID: PMC3426632  PMID: 22278412
viral vector gene transfer; Sindbis; axon regeneration
18.  Chemical genetic-mediated spatial regulation of protein expression in neurons reveals an axonal function for WldS 
Chemistry & biology  2012;19(2):179-187.
The degeneration of axons is the underlying pathological process of several neurological disorders. The Wallerian degeneration (WldS) slow protein, which is primarily nuclear, markedly inhibits axonal degeneration. Contradictory models have been proposed to explain its mechanism, including a role in the nucleus where it affects gene transcription, and roles outside the nucleus where it regulates unknown effectors. To determine which pool of WldS accounts for its axon protective effects, we developed a strategy to control the spatial expression of proteins within neurons. This strategy couples a chemical genetic method to control protein stability with microfluidic culturing. Using neurons that are selectively deficient in WldS in axons, we show that the axonal pool of WldS is necessary for protection from axon degeneration. These results implicate an axonal pathway regulated by WldS that controls axon degeneration.
PMCID: PMC3292772  PMID: 22365601
19.  A detection of unexpected blood antibody at the time of transfusion was needed, during the operation -A case report- 
To avoid the occurrence of fatal complications of blood transfusion, several tests are implemented before transfusion. The tests include ABO typing, Rh typing, cross-matching test and blood antibody screening test, and in usual they are completed before transfusion. However in the case of repetitive operations done via brief distance, reexamination for blood antibody tends to be omitted. After 2 previous operations, 30 years old male patient showed positive blood antibody screening during the third operation. Although antibody screening test performed before the first operation, no unexpected blood antibody was detected. During the third operation, after the decision to start transfusion was made, it took two hours to find appropriate blood. There was no significant deterioration of patient's condition but the loss of time could lead to critical consequences. We present this case to make anesthesiologists and surgeons aware of possibility of unexpected blood antibody detection after transfusion.
PMCID: PMC3558653  PMID: 23372889
Antibody; Complication; Transfusion
20.  In Vitro Culture-Induced Pluripotency of Human Spermatogonial Stem Cells 
BioMed Research International  2012;2013:143028.
Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotency in vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs in in vitro cultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2–4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs during in vitro culture.
PMCID: PMC3591227  PMID: 23484080
21.  Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity 
Journal of medicinal chemistry  2011;54(21):7535-7546.
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring non-polar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of non-polar interactions governing GCPII affinity towards novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. Based on those data, we designed, synthesized and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
PMCID: PMC3222833  PMID: 21923190
PSMA; NAALADase; GCPII; zinc peptidase; folate hydrolase; inhibition; quantum mechanics/molecular mechanics (QM/MM)
22.  Examination of Axonal Injury and Regeneration in Microfluidic Neuronal Culture Using Pulsed Laser Microbeam Dissection 
Lab on a Chip  2010;10(16):2083-2092.
We describe the integrated use of pulsed laser microbeams and microfluidic cell culture to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, λ=532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 μm and 50 μm in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1–2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interfere with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a microfluidic cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration.
PMCID: PMC3380453  PMID: 20532390
24.  Analysis of prognostic factors and 5-year survival rate in patients with hepatocellular carcinoma: a single-center experience 
Hepatocellular carcinoma (HCC), which is the third most common cancer in Korea, has a very poor prognosis. However, only a few studies have performed a comprehensive survival-related analysis in all patients who were consecutively diagnosed and treated over a given period of time. The aim of this study was to determine the 5-year survival rate and its prognostic factors among HCC patients.
In total, 257 patients who were consecutively diagnosed with HCC between January 2000 and December 2003 were followed until death or until December 2008. We analyzed their survival outcomes according to their clinical characteristics, tumor staging, and treatment modalities, and determined the independent prognostic factors affecting survival.
The patients were aged 59±10 years (mean±SD). During the follow-up period, 223 patients (86.8%) died and the overall median survival was 10.8 months; the 1-, 3-, and 5-year survival rates were 44.4%, 21.0%, and 12.1%, respectively. The outcomes in patients with tumor node metastasis (TNM) stage I or II and Child-Pugh class A or B were significantly better with surgical resection than with other treatment modalities (P<0.01). Patients who underwent supplementary transcatheter arterial chemoembolization as a second-line treatment after surgical resection had better outcomes than those who underwent surgical resection alone (P=0.02). Initial symptoms, Child-Pugh class, serum alpha-fetoprotein, tumor size, portal vein thrombosis, and TNM stage were found to be independent prognostic factors for survival among HCC patients.
This retrospective cohort study elucidated survival outcomes and prognostic factors affecting survival in HCC patients at a single center.
PMCID: PMC3326996  PMID: 22511903
Hepatocellular carcinoma; Survival; Prognosis; Treatment; Tumor staging
25.  The Frequency and Risk Factors of Colorectal Adenoma in Health-Check-up Subjects in South Korea: Relationship to Abdominal Obesity and Age 
Gut and Liver  2010;4(1):36-42.
Obesity is associated with the risk of colorectal cancer. However, there is a lack of information about the relationship between obesity and colorectal adenoma. We investigated whether general and abdominal obesity are risk factors for colorectal adenoma.
Subjects who received health check-ups, including colonoscopy, from April 2006 to September 2007 in Chung-Ang University Hospital were included (n=1,316). The frequency and characteristics of colorectal adenomas were analyzed according to demographic features, past history, blood tests, body mass index, and components of metabolic syndrome. Abdominal obesity was defined as a waist circumference of ≥80 cm in women and ≥90 cm in men.
The sex ratio of the subjects was 1.9:1 (male:female) and their age was 47.7±10.0 years (mean±SD). In univariate analysis, abdominal obesity was significantly associated with the frequency of colorectal adenoma (26.5% "yes" vs 16.9% "no"; p<0.001). The frequency of colorectal adenoma was significantly higher among males, older patients, current smokers, and subjects with fasting hyperglycemia (≥100 mg/dL) or fatty liver (p<0.05). Multivariate analysis identified that male sex (odds ratio [OR], 1.5; 95% confidence interval [CI], 1.0-2.2), old age (age ≥60 years; OR, 6.7; 95% CI, 3.5-12.5), and abdominal obesity (OR, 1.5; 95% CI, 1.0-2.2) were independent risk factors for colorectal adenoma (p<0.05). The frequency of multiple adenomas (more than two sites) was also significantly higher in subjects with abdominal obesity. However, the effect of abdominal obesity on the development of colorectal adenoma decreased in elderly people.
Abdominal obesity is an independent risk factor for colorectal adenoma and its multiplicity, especially in younger people in South Korea.
PMCID: PMC2871608  PMID: 20479911
Abdominal obesity; Colorectal adenoma

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