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1.  High TOP2B/TOP2A expression ratio at diagnosis correlates with favourable outcome for standard chemotherapy in acute myeloid leukaemia 
British Journal of Cancer  2012;107(1):108-115.
Background:
Cytosine arabinoside-based chemotherapy coupled with anthracycline is currently the first-line treatment for acute myeloid leukaemia (AML), but diverse responses to the regimen constitute obstacles to successful treatment. Therefore, outcome prediction to chemotherapy at diagnosis is believed to be a critical consideration.
Methods:
The mRNA expression of 12 genes closely involved in the actions of cytosine arabinoside and anthracycline was evaluated by real-time reverse transcriptase PCR (RT–PCR), in 54 diagnostic bone marrow specimens of M2-subtype AML.
Results:
Low expression levels of ribonucleotide reductase M2 (RRM2) and high expression levels of topoisomerase 2 beta (TOP2B) were correlated with longer survival in a univariate analysis. Another interesting finding is that high ratios of TOP2B/RRM2 and TOP2B/TOP2 alpha (TOP2A) in a combined analysis were also shown to have a prognostic impact for longer survival with improved accuracy. Among the four markers, when adjusted for the influence of other clinical factors in multivariate analysis, the TOP2B/TOP2A ratio was significantly correlated with treatment outcomes; patients with high ratios trended toward longer disease-free survival (HR, 0.24; P=0.002) and overall survival (HR, 0.29; P=0.005).
Conclusion:
Genes with distinct expression profiles such as TOP2B/TOP2A expression ratio at diagnosis can be employed for outcome prediction after the treatment with standard regimens in AML patients with M2 subtype.
doi:10.1038/bjc.2012.206
PMCID: PMC3389410  PMID: 22627319
AML; standard chemotherapy; prognosis; topoisomerase 2
2.  Tumour-infiltrating CD8+ lymphocytes as an independent predictive factor for pathological complete response to primary systemic therapy in breast cancer 
British Journal of Cancer  2013;109(10):2705-2713.
Background:
Tumour-infiltrating lymphocytes (TILs) are known to be associated with response to primary systemic therapy (PST) in breast cancer. This study was conducted to assess the association of TIL subsets with pathological complete response (pCR) after PST in breast cancer in relation to breast cancer subtype, breast cancer stem cell (BCSC) phenotype and epithelial–mesenchymal transition (EMT).
Methods:
The pre-chemotherapeutic biopsy specimens of 153 breast cancer patients who underwent surgical resection after anthracycline- or anthracycline/taxane-based PST were analysed. TIL subsets (CD4+, CD8+, and FOXP3+ TILs), BCSC phenotype, and the expression of EMT markers were evaluated by immunohistochemistry and were correlated with pCR after PST.
Results:
Infiltration of CD4+ and CD8+ T lymphocytes was closely correlated with BCSC phenotype and EMT. High levels of CD4+, CD8+, and FOXP3+ TILs were associated with pCR, and CD8+ TILs were found to be an independent predictive factor for pCR. In addition, CD8+ TILs were associated with pCR irrespective of breast cancer subtype, CD44+/CD24− phenotype, EMT, and chemotherapeutic regimen in subgroup analyses.
Conclusion:
These findings indicate that CD8+ cytotoxic T lymphocytes are a key component of TILs associated with chemo-response and can be used as a reliable predictor of response to anthracycline- or anthracycline/taxane-based PST in breast cancer.
doi:10.1038/bjc.2013.634
PMCID: PMC3833219  PMID: 24129232
primary systemic therapy; pathological complete response; tumour-infiltrating lymphocytes; CD8; cytotoxic T lymphocyte
3.  The effect of resveratrol on normal and osteoarthritic chondrocyte metabolism 
Bone & Joint Research  2014;3(3):51-59.
Background
Resveratrol is a polyphenolic compound commonly found in the skins of red grapes. Sirtuin 1 (SIRT1) is a human gene that is activated by resveratrol and has been shown to promote longevity and boost mitochondrial metabolism. We examined the effect of resveratrol on normal and osteoarthritic (OA) human chondrocytes.
Methods
Normal and OA chondrocytes were incubated with various concentrations of resveratrol (1 µM, 10 µM, 25 µM and 50 µM) and cultured for 24, 48 or 72 hours or for six weeks. Cell proliferation, gene expression, and senescence were evaluated.
Results
SIRT1 was significantly upregulated in normal chondrocytes with resveratrol concentrations of 25 µM and 50 µM on both two- (2D) (both p = 0.001) and three-dimensional (3D) cultures (p = 0.008 and 0.001, respectively). It was significantly upregulated in OA chondrocytes treated with 10 µM, 25 µM and 50 µM resveratrol on 2D cultures (p = 0.036, 0.002 and 0.001, respectively) and at 50 µM concentration on 3D cultures (p = 0.001). At 72 hours, the expression of collagen (COL)-10, aggrecan (AGG), and runt-related transcription factor 2 (RUNX2) was significantly greater in both 25 µM (p = 0.011, 0.006 and 0.015, respectively) and 50 µM (p = 0.019, 0.004 and 0.002, respectively) resveratrol-treated normal chondrocyte cultures. In OA chondrocytes, expression of COL10 and RUNX2 was significantly greater in 25 µM (p = 0.004 and 0.024) and 50 µM (p = 0.004 and 0.019) cultures at 72 hours on 3D cultures.
Conclusions
At concentrations of 25 µM and/or 50 µM, resveratrol treatment significantly upregulates SIRT1 gene expression in normal and osteoarthritic chondrocytes. Resveratrol induces chondrocytes into a hypertrophic state through upregulation of COL1, COL10, and RUNX2.
Cite this article: Bone Joint Res 2014;3:51–9.
doi:10.1302/2046-3758.33.2000226
PMCID: PMC3963380  PMID: 24615710
Resveratrol; SIRT1; Osteoarthritis; Chondrocyte; Longevity; Metabolism
4.  Correlation of the apparent diffusion coefficiency values on diffusion-weighted imaging with prognostic factors for breast cancer 
The British Journal of Radiology  2012;85(1016):e474-e479.
Objective
The aim of this study was to correlate the apparent diffusion coefficient (ADC) value of breast cancer with prognostic factors.
Methods
335 patients with invasive ductal carcinoma not otherwise specified (IDC NOS) and ductal carcinoma in situ (DCIS) who underwent breast MRI with diffusion-weighted imaging were included in this study. ADC of breast cancer was calculated using two b factors (0 and 1000 s mm–2). Mean ADCs of IDC NOS and DCIS were compared and evaluated. Among cases of IDC NOS, mean ADCs were compared with lymph node status, size and immunochemical prognostic factors using Student's t-test. ADC was also correlated with histological grade using the Kruskal–Wallis test.
Results
Mean ADC of IDC NOS was significantly lower than that of DCIS (p<0.001). However, the mean ADC of histological grade of IDC NOS was not significantly different (p=0.564). Mean ADC of oestrogen receptor (ER)-positive or progesterone receptor (PR)-positive cancer was significantly lower than that of ER-negative or PR-negative cancer (p=0.003 vs p=0.032). Mean ADC of Ki-67 index-positive cancer was significantly lower than that of Ki-67 index-negative cancer (p=0.028). Mean ADC values of cancers with increased microvascular density (MVD) were significantly lower than those of cancer with no MVD increase (p=0.009). No correlations were observed between mean ADC value and human growth factor receptor 2 expression, tumour size and lymph node metastasis.
Conclusion
Low ADC value was correlated with positive expression of ER, PR, increased Ki-67 index, and increased MVD of breast cancer.
doi:10.1259/bjr/79381464
PMCID: PMC3587081  PMID: 22128125
5.  BRCA1 negatively regulates IGF-1 expression through an estrogen-responsive element-like site 
Cell Death & Disease  2012;3(6):e336-.
The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is critical for both normal mammary gland development and malignant transformation. It has been reported that the IGF-1 stimulates breast cancer cell proliferation and is upregulated in tumors with BRCA1/2 mutations. We report here that IGF-1 is negatively regulated by BRCA1 at the transcriptional level in human breast cancer cells. BRCA1 knockdown (BRCA1-KD) induces the expression of IGF-1 mRNA in MCF7 cells in an estrogen receptor α (ERα)-dependent manner. We found that both BRCA1 and ERα bind to the endogenous IGF-1 promoter region containing an estrogen-responsive element-like (EREL) site. BRCA1-KD does not significantly affect ERα binding on the IGF-1 promoter. Reporter analysis demonstrates that BRCA1 could regulate IGF-1 transcripts via this EREL site. In addition, enzyme-linked immunosorbent assay revealed that de-repression of IGF-1 transcription by BRCA1-KD increases the level of extracellular IGF-1 protein, and secreted IGF-1 seems to increase the phospho-IGF-1Rβ and activate its downstream signaling pathway. Blocking the IGF-1/IGF-1R/phosphoinositide 3-kinase (PI3K)/AKT pathway either by a neutralizing antibody or by small-molecule inhibitors preferentially reduces the proliferation of BRCA1-KD cells. Furthermore, the IGF-1-EREL-Luc reporter assay demonstrates that various inhibitors, which can inhibit the IGF-1R pathway, can suppress this reporter activity. These findings suggest that BRCA1 defectiveness keeps turning on IGF-1/PI3K/AKT signaling, which significantly contributes to increase cell survival and proliferation.
doi:10.1038/cddis.2012.78
PMCID: PMC3388245  PMID: 22739988
BRCA1; IGF-1; negative regulation; ERα; positive-feedback activation
6.  c-Myc expression is related with cell proliferation and associated with poor clinical outcome in human gastric cancer. 
Journal of Korean Medical Science  1999;14(5):526-530.
We underwent protein assay for Myc expression in 76 human gastric cancer tissues using immunohistochemistry. Expression of Myc protein was analyzed according to proliferative indices measured by flow cytometry. Levels of Myc protein expression was evaluated by correlating with biologic and clinical parameters. In 36 (47.4%) of 76 primary gastric cancers, overexpression of Myc was observed. We could observe expression of Myc protein in a significant portion of early gastric cancer (42.9%). Expression of Myc protein was demonstrated to be more frequent in poorly differentiated cancer cells (p=0.043). However, expression of Myc protein had little influence over progress or extent of the disease. Expression of Myc protein was significantly correlated with increased proliferative activity (p=0.032) and patients with high levels of Myc expression had poor disease-free survival. In a certain proportion of human gastric cancer, Myc protein may function as a regulator of cancer cell growth and expression of Myc may represent an aggressive phenotype of gastric cancer.
PMCID: PMC3054460  PMID: 10576148
7.  The first imported case of pulmonary coccidioidomycosis in Korea. 
Journal of Korean Medical Science  1999;14(2):206-209.
Coccidioidomycosis is an endemic disease found in the southwestern part of North America. Travellers who visit the endemic area may carry the infection. We report a case of pulmonary coccidioidomycosis in a 74-year-old woman. She was healthy before visiting Arizona, U.S.A twice. After returning home, she began to complain of intermittent dry coughing. The symptom was mild, however, and she was treated symptomatically. Later a chest radiograph, which was taken 4 years after the onset of the symptom, showed a solitary pulmonary nodule in the right upper lobe. By percutaneous needle aspiration, a few clusters of atypical cells were noted in the necrotic background. A right upper and middle lobectomy was done. A 1.5 x 1.5 x 1.2 cm sized tan nodule was present in otherwise normal lung parenchyma. Microscopically, the nodule consisted of aggregates of multiple solid granulomas inside of which was mostly necrotic. Neutrophils and nuclear debris were scattered along the periphery of the necrotic foci. Numerous multinucleated giant cells were associated with the granulomas. In the necrotic area, mature spherules of Coccidioides immitis, which were 30-100 microm in diameter, were present. They contained numerous endospores which ranged from 5 to 15 microm and were also noted in multinucleated giant cells. The diagnosis of coccidioidomycosis was made. She is doing well after the resection.
PMCID: PMC3054355  PMID: 10331569
8.  Mixed hepatoblastoma in an adult--a case report and literature review. 
Journal of Korean Medical Science  1997;12(4):369-373.
Hepatoblastoma is thought to originate from embryonal hepatic tissue, and most of these tumors occur in children under the age of 2 years. Hepatoblastoma in adults is extremely rare, and the prognosis is much worse than the mixed hepatoblastoma of childhood. We experienced a case of mixed hepatoblastoma in a 51 year old female patient. She had been suffering from a mild pain and a palpable lump in the epigastric area. Serum AFP was 43,850 ng/ml. Computerized tomography and selective abdominal angiography showed a large low-density mass. With a suspicion of hepatocellular carcinoma of the left lobe, a left lateral segmentectomy was performed. The external surface showed a huge protruding mass and the capsule was previously ruptured. On section, the tumor was a 11 x 7 cm sized expanding mass which had a variegated surface composed of yellow-white friable tissue with multifocal hemorrhagic areas. Microscopic examination revealed a tumor consisted of epithelial and mesenchymal elements. The mesenchymal cells were spindle in shape and proliferated over the whole tumor with focal osteosarcomatous differentiation. The epithelial components showed well-differentiated hepatocellular carcinoma-like areas, poorly differentiated acinar or tubular structures.
PMCID: PMC3054210  PMID: 9288639
9.  Spinal congenital dermal sinus--experience of 5 cases over a period of 10 years. 
Spinal congenital dermal sinus (CDS) is a rare entity which supposedly results from the failure of the neuroectoderm to separate from the cutaneous ectoderm during the process of neurulation. The lesions are most frequent at the lumbosacral followed by the occipital region. CDS of the thoracic region is very rare. The patients with spinal CDS present with meningitis and/or mass effect from the associated inclusion tumor. They are usually dermoid or epidermoid cysts. Teratoma is rarely associated. The authors experienced 5 cases of spinal CDS over a 10 year period. Of the 5 cases, 2 were at thoracic and 3 were at lumbosacral levels. Dermoid cyst, epidermoid cyst and teratoma were associated in one case each. Two cases presented with neurological deficit and meningitis while an additional case presented with neurological deficit and a history of probable meningitis. Pain was present in 2 cases. Magnetic resonance imaging played an important role in the diagnosis of the lesion and planning of surgery. All the cases showed a good response to surgery even though one patient had persistent neurological deficit.
PMCID: PMC3053715  PMID: 8305142
10.  Activated STING in a Vascular and Pulmonary Syndrome 
The New England journal of medicine  2014;371(6):507-518.
BACKGROUND
The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation.
METHODS
We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls.
RESULTS
We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mono-nuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibro-blasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was reduced by JAK inhibitors.
CONCLUSIONS
STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173.
doi:10.1056/NEJMoa1312625
PMCID: PMC4174543  PMID: 25029335
11.  Diffusion-Weighted Imaging of Orbital Masses: Multi-Institutional Data Support a 2-ADC Threshold Model to Categorize Lesions as Benign, Malignant, or Indeterminate 
BACKGROUND AND PURPOSE
DWI has been increasingly used to characterize orbital masses and provides quantitative information in the form of the ADC, but studies of DWI of orbital masses have shown a range of reported sensitivities, specificities, and optimal threshold ADC values for distinguishing benign from malignant lesions. Our goal was to determine the optimal use of DWI for imaging orbital masses through aggregation of data from multiple centers.
MATERIALS AND METHODS
Source data from 3 previous studies of orbital mass DWI were aggregated, and additional published data points were gathered. Receiver operating characteristic analysis was performed to determine the sensitivity, specificity, and optimal ADC thresholds for distinguishing benign from malignant masses.
RESULTS
There was no single ADC threshold that characterized orbital masses as benign or malignant with high sensitivity and specificity. An ADC of less than 0.93 × 10−3 mm2/s was more than 90% specific for malignancy, and an ADC of less than 1.35 × 10−3 mm2/s was more than 90% sensitive for malignancy. With these 2 thresholds, 33% of this cohort could be characterized as “likely malignant,” 29% as “likely benign,” and 38% as “indeterminate.”
CONCLUSIONS
No single ADC threshold is highly sensitive and specific for characterizing orbital masses as benign or malignant. If we used 2 thresholds to divide these lesions into 3 categories, however, a majority of orbital masses can be characterized with >90% confidence.
doi:10.3174/ajnr.A3619
PMCID: PMC4138308  PMID: 23868150
12.  Gadoxetic acid-enhanced MRI for T-staging of gallbladder carcinoma: emphasis on liver invasion 
The British Journal of Radiology  2013;87(1033):20130608.
Objective:
To evaluate the diagnostic performance of gadoxetic acid-enhanced MRI with an emphasis on the usefulness of the hepatobiliary phase (HBP) in T-staging of gallbladder carcinoma.
Methods:
66 patients with surgically confirmed gallbladder carcinoma underwent MRI. Two radiologists independently reviewed two sets of gadoxetic acid-enhanced MRI without and with the HBP. Local tumour spread was evaluated according to T-staging, and the results were compared with pathological findings. The diagnostic performance of two image sets to differentiate each T-stage was compared.
Results:
The sensitivities of MRI with the HBP to differentiate T1 vs ≥T2 lesions, ≤T2 vs ≥T3 lesions and ≤T3 vs T4 lesions were 96.3%, 85.7% and 100% for Observer 1 and 92.6%, 95.2% and 100% for Observer 2, respectively (p < 0.0001). By adding the HBP, the sensitivities to differentiate ≤T2 vs ≥T3 lesions were increased from 66.7% to 85.7% for Observer 1 and from 81.0% to 95.2% for Observer 2, although there was no significant difference (p > 0.05). The overall accuracies for T-staging were increased from 80.3% to 86.4% for Observer 1, a statistically significant degree (p = 0.046), and from 83.8% to 87.9% for Observer 2 (p > 0.05). The k-value for the two observers indicated excellent agreement.
Conclusion:
Gadoxetic acid-enhanced MRI provided acceptable diagnostic performance for T-staging of gallbladder carcinoma. Addition of the HBP aids in the detection of liver invasion.
Advances in knowledge:
In the T-staging of gallbladder carcinoma, gadoxetic acid-enhanced MRI with the HBP may enhance detection of liver invasion.
doi:10.1259/bjr.20130608
PMCID: PMC3898979  PMID: 24288397
13.  Neuritin can normalize neural deficits of Alzheimer's disease 
Cell Death & Disease  2014;5(11):e1523-.
Reductions in hippocampal neurite complexity and synaptic plasticity are believed to contribute to the progressive impairment in episodic memory and the mild cognitive decline that occur particularly in the early stages of Alzheimer's disease (AD). Despite the functional and therapeutic importance for patients with AD, intervention to rescue or normalize dendritic elaboration and synaptic plasticity is scarcely provided. Here we show that overexpression of neuritin, an activity-dependent protein, promoted neurite outgrowth and maturation of synapses in parallel with enhanced basal synaptic transmission in cultured hippocampal neurons. Importantly, exogenous application of recombinant neuritin fully restored dendritic complexity as well as spine density in hippocampal neurons prepared from Tg2576 mice, whereas it did not affect neurite branching of neurons from their wild-type littermates. We also showed that soluble recombinant neuritin, when chronically infused into the brains of Tg2576 mice, normalized synaptic plasticity in acute hippocampal slices, leading to intact long-term potentiation. By revealing the protective actions of soluble neuritin against AD-related neural defects, we provide a potential therapeutic approach for patients with AD.
doi:10.1038/cddis.2014.478
PMCID: PMC4260736  PMID: 25393479
14.  Mapping of two suppressors of OVATE (sov) loci in tomato 
Heredity  2013;111(3):256-264.
Tomato fruit shape varies significantly in the cultivated germplasm. To a large extent, this variation can be explained by four genes including OVATE. While most varieties with the OVATE mutation bear elongated fruits, some accessions carry round fruit, suggesting the existence of suppressors of OVATE in the germplasm. We developed three intraspecific F2 populations with parents that carried the OVATE mutation but differed in fruit shape. We used a bulk segregant analysis approach and genotyped the extreme classes using a high-throughput genotyping platform, the SolCAP Infinium Assay. The analyses revealed segregation at two quantitative trait loci (QTLs), sov1 and sov2. These loci were confirmed by genotyping and QTL analyses of the entire population. More precise location of those loci using progeny testing confirmed that sov1 on chromosome 10 controlled obovoid and elongated shape, whereas sov2 on chromosome 11 controlled mainly elongated fruit shape. Both loci were located in intervals of <2.4 Mb on their respective chromosomes.
doi:10.1038/hdy.2013.45
PMCID: PMC3746822  PMID: 23673388
BSA; fruit morphology; SNP array;  Solanum lycopersicum ; pear shape; progeny test
15.  GCPII Imaging and Cancer 
Current medicinal chemistry  2012;19(9):1346-1359.
Glutamate carboxypeptidase II (GCPII) in the central nervous system is referred to as the prostate-specific membrane antigen (PSMA) in the periphery. PSMA serves as a target for imaging and treatment of prostate cancer and because of its expression in solid tumor neovasculature has the potential to be used in this regard for other malignancies as well. An overview of GCPII/PSMA in cancer, as well as a discussion of imaging and therapy of prostate cancer using a wide variety of PSMA-targeting agents is provided.
PMCID: PMC4076792  PMID: 22304713
Prostate-specific membrane antigen; PSMA; prostate cancer; molecular imaging; PET; SPECT
16.  hnRNP Q regulates translation of p53 in normal and stress conditions 
Cell Death and Differentiation  2012;20(2):226-234.
The responses to numerous stress signals are important for cellular growth and survival. The p53 tumor-suppressor protein is stabilized under stress conditions and induces transcription of several genes to regulate cell cycle and apoptosis. Regarding p53 protein accumulation, inhibition of proteasomal degradation of p53 protein, which is mainly mediated by Mdm2, has received much attention. Here, we demonstrate that regulation of translation initiation is also crucial for p53 protein accumulation. Furthermore, we report that heterogeneous nuclear ribonucleoprotein (hnRNP) Q binds to the 5′-untranslated region (UTR) of mouse p53 mRNA and regulates translation efficiency of p53 and apoptosis progression. We also suggest that changes in cytosolic hnRNP Q levels contribute to cell cycle-dependent translational differences in p53 mRNA.
doi:10.1038/cdd.2012.109
PMCID: PMC3554343  PMID: 22935615
hnRNP Q; IRES; p53; translation
17.  Mycobacterial HBHA induces endoplasmic reticulum stress-mediated apoptosis through the generation of reactive oxygen species and cytosolic Ca2+ in murine macrophage RAW 264.7 cells 
Cell Death & Disease  2013;4(12):e957-.
Mycobacterial heparin-binding haemagglutinin antigen (HBHA) is a virulence factor that induces apoptosis of macrophages. Endoplasmic reticulum (ER) stress-mediated apoptosis is an important regulatory response that can be utilised to study the pathogenesis of tuberculosis. In the present study, HBHA stimulation induced ER stress sensor molecules in a caspase-dependent manner. Pre-treatment of RAW 264.7 cells with an IκB kinase 2 inhibitor reduced not only C/EBP homology protein expression but also IL-6 and monocyte chemotactic protein-1 (MCP-1) production. BAPTA-AM reduced both ER stress responses and caspase activation and strongly suppressed HBHA-induced IL-6 and MCP-1 production in RAW 264.7 cells. Enhanced reactive oxygen species (ROS) production and elevated cytosolic [Ca2+]i levels were essential for HBHA-induced ER stress responses. Collectively, our data suggest that HBHA induces cytosolic [Ca2+]i, which influences the generation of ROS associated with the production of proinflammatory cytokines. These concerted and complex cellular responses induce ER stress-associated apoptosis during HBHA stimulation in macrophages. These results indicate that the ER stress pathway has an important role in the HBHA-induced apoptosis during mycobacterial infection.
doi:10.1038/cddis.2013.489
PMCID: PMC3877560  PMID: 24336077
ER stress; macrophage; mycobacteria
18.  Identification of differentially expressed genes according to chemosensitivity in advanced ovarian serous adenocarcinomas: expression of GRIA2 predicts better survival 
British Journal of Cancer  2012;107(1):91-99.
Background:
The purpose of this study was to identify genes that are differentially expressed in chemosensitive serous papillary ovarian carcinomas relative to those expressed in chemoresistant tumours.
Methods:
To identify novel candidate biomarkers, differences in gene expression were analysed in 26 stage IIIC/IV serous ovarian adenocarcinomas (12 chemosensitive tumours and 14 chemoresistant tumours). We subsequently investigated the immunohistochemical expression of GRIA2 in 48 independent sets of advanced ovarian serous carcinomas.
Results:
Microarray analysis revealed a total of 57 genes that were differentially expressed in chemoresistant and chemosensitive tumours. Of the 57 genes, 39 genes were upregulated and 18 genes were downregulated in chemosensitive tumours. Five differentially expressed genes (CD36, LIFR, CHL1, GRIA2, and FCGBP) were validated by quantitative real-time PCR. The expression of GRIA2 was validated at the protein level by immunohistochemistry, and patients with GRIA2 expression showed a longer progression-free and overall survival (P=0.051 and P=0.031 respectively).
Conclusions:
We found 57 differentially expressed genes to distinguish between chemosensitive and chemoresistant tumours. We also demonstrated that the expression of GRIA2 among the differentially expressed genes provides better prognosis of patients with advanced serous papillary ovarian adenocarcinoma.
doi:10.1038/bjc.2012.217
PMCID: PMC3389416  PMID: 22644307
gene expression profiling; microarray; ovarian serous adenocarcinoma; GRIA2; survival
19.  Hypothalamic ATF3 is involved in regulating glucose and energy metabolism in mice 
Diabetologia  2013;56(6):1383-1393.
Aims/hypothesis
The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study.
Methods
We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp).
Results
Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription.
Conclusions/interpretation
Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp.
Electronic supplementary material
The online version of this article (doi:10.1007/s00125-013-2879-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
doi:10.1007/s00125-013-2879-z
PMCID: PMC3648686  PMID: 23462798
Agrp; ATF3; Energy expenditure; Food intake; FoxO1; Hypothalamus
20.  Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin 
Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca2+]i through G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca2+]i and are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+ bowl and the regulator of K+ conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+ bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons.
doi:10.1155/2013/323709
PMCID: PMC3638619  PMID: 23662129
21.  Aligned Silk-Based 3D Architectures for Contact Guidance in Tissue Engineering 
Acta Biomaterialia  2011;8(4):1530-1542.
An important challenge in the biomaterials field is to mimic the structure of functional tissues via cell and extracellular matrix (ECM) alignment and anisotropy. Toward this goal, silk-based scaffolds resembling bone lamellar structure were developed using a freeze-drying technique. The structure could be controlled directly by solute concentration and freezing parameters, resulting in lamellar scaffolds with regular morphology. Different post-treatments were investigated to induce water stability, such as methanol, water annealing and steam sterilization. The resulting structures exhibited significant differences in terms of morphological integrity, structure and mechanical properties. the lamellar thicknesses were around ~2,6 μm for the methanol treated scaffolds and ~5,8 μm for water-annealed. These values are in the range of the reported for human lamellar bone. Human bone marrow-derived mesenchymal stem cells (hMSCs) were seeded on these silk fibroin lamellar scaffolds and grown under osteogenic conditions to assess the effect of the microstructure on cell behaviour. Collagen in the newly deposited ECM, was found aligned along the lamellar architectures. In the case of methanol treated lamellar structures the hMSCs were able to migrate into the interior of the scaffolds producing a multilamellar hybrid construct. The present morphology constitutes a useful pattern onto which hMSCs cells attach and proliferate for guided formation of a highly oriented extracellular matrix.
doi:10.1016/j.actbio.2011.12.015
PMCID: PMC3289723  PMID: 22202909
Silk fibroin; scaffold; freeze-drying; directional freezing; tissue engineering; lamellar morphology; cell alignment
22.  Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis 
A series of in vitro studies were carried out to determine i) the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii) the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS) in in vitro experiments. Untreated corn (C) and enzyme-treated corn (EC) were combined with soy bean meal with (ES) and without (S) enzyme treatment or formaldehyde treatment (FS). Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS) with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM) digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS). The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity.
doi:10.5713/ajas.2012.12507
PMCID: PMC4093482  PMID: 25049798
Enzyme; Formaldehyde; Synchronicity; Rumen Fermentation; Microbial Protein Synthesis
23.  A critical step for JNK activation: isomerization by the prolyl isomerase Pin1 
Cell Death and Differentiation  2011;19(1):153-161.
c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1−/− cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.
doi:10.1038/cdd.2011.82
PMCID: PMC3252824  PMID: 21660049
c-Jun N-terminal kinase; peptidyl-prolyl cis/trans-isomerase; apoptosis
24.  A Six-Month Supplementation of Mulberry, Korean Red Ginseng, and Banaba Decreases Biomarkers of Systemic Low-Grade Inflammation in Subjects with Impaired Glucose Tolerance and Type 2 Diabetes 
We sought the long-term efficacy of traditionally used antidiabetic herbs in controlling blood glucose homeostasis and low-grade inflammation. Ninety-four subjects with either impaired glucose tolerance or mild T2D were randomized either to treatment arm or placebo arm and received 1 : 1 : 1 mixture of ginseng roots, mulberry leaf water extract, and banaba leaf water extract (6 g/d) for 24 weeks. Oral 75 g glucose tolerance test was performed to measure glucose and insulin responses. Blood biomarkers of low-grade inflammation were also determined. Results found no significant difference in glucose homeostasis control measure changes. However, plasma intracellular adhesion molecule-1 (ICAM-1) concentration was decreased showing a significant between-treatment changes (P = 0.037). The concentrations of vascular cell adhesion molecule-1 (VCAM-1) (P = 0.014) and ICAM-1 (P = 0.048) were decreased in the treatment group at week 24, and the oxidized low-density lipoprotein (ox-LDL) concentration was reduced at week 24 compared to the baseline value in the treatment group (P = 0.003). These results indicate a long-term supplementation of ginseng, mulberry leaf, and banaba leaf suppresses inflammatory responses in T2D.
doi:10.1155/2012/735191
PMCID: PMC3303547  PMID: 22474520
25.  Familial neuromyelitis optica 
Neurology  2010;75(4):310-315.
Background:
Detection of aquaporin-4–specific immunoglobulin G (IgG) has expanded the spectrum of neuromyelitis optica (NMO). Rare reports of familial aggregation have suggested a component of genetic susceptibility but these reports mostly antedated the discovery of the NMO-IgG biomarker and recently updated diagnostic criteria.
Methods:
We report a case series describing the demographic, clinical, neuroimaging, and NMO-IgG serologic status of 12 multiplex NMO pedigrees with a total of 25 affected individuals.
Results:
Twenty-one patients (84%) were women. Families were Asian (n = 5), Latino (n = 4), white (n = 1), or African (n = 2). Apparent transmission was either maternal (n = 5) or paternal (n = 2). In 1 family, 3 individuals had NMO; in the others, 2 individuals were affected. Sibling pairs (n = 6), parent-child (n = 4), and aunt-niece (n = 3) pairs were observed. Nineteen patients (76%) were NMO-IgG positive. Twelve (48%) had clinical or serologic evidence of another autoimmune disease. Familial occurrence of NMO occurs in approximately 3% of patients with well-established diagnosis of NMO.
Conclusions:
A small proportion of patients with NMO have relatives with this condition, but familial occurrence is more common than would be expected from its frequency in the general population. Familial NMO is indistinguishable from sporadic NMO based on clinical symptoms, age at onset, sex distribution, and frequency of NMO-IgG detection. One or 2 generations were affected and affected individuals represented a small fraction of family members. Taken together, these data suggest complex genetic susceptibility in NMO.
GLOSSARY
= aquaporin-4;
= confidence interval;
= human leukocyte antigen;
= immunoglobulin G;
= longitudinally extensive transverse myelitis;
= multiple sclerosis;
= neuromyelitis optica;
= optic neuritis;
= odds ratio.
doi:10.1212/WNL.0b013e3181ea9f15
PMCID: PMC2918892  PMID: 20660861

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