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1.  Reduced Expression of Galectin-9 Contributes to a Poor Outcome in Colon Cancer by Inhibiting NK Cell Chemotaxis Partially through the Rho/ROCK1 Signaling Pathway 
PLoS ONE  2016;11(3):e0152599.
Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearman's rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearman's rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56+ NK cell infiltration (R2 = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.
doi:10.1371/journal.pone.0152599
PMCID: PMC4814049  PMID: 27028892
2.  Systolic MOLLI T1 mapping with heart-rate-dependent pulse sequence sampling scheme is feasible in patients with atrial fibrillation 
Background
T1 mapping enables assessment of myocardial characteristics. As the most common type of arrhythmia, atrial fibrillation (AF) is often accompanied by a variety of cardiac pathologies, whereby the irregular and usually rapid ventricle rate of AF may cause inaccurate T1 estimation due to mis-triggering and inadequate magnetization recovery. We hypothesized that systolic T1 mapping with a heart-rate-dependent (HRD) pulse sequence scheme may overcome this issue.
Methods
30 patients with AF and 13 healthy volunteers were enrolled and underwent cardiovascular magnetic resonance (CMR) at 3 T. CMR was repeated for 3 patients after electric cardioversion and for 2 volunteers after lowering heart rate (HR). A Modified Look-Locker Inversion Recovery (MOLLI) sequence was acquired before and 15 min after administration of 0.1 mmol/kg gadopentetate dimeglumine. For AF patients, both the fixed 5(3)3/4(1)3(1)2 and the HRD sampling scheme were performed at diastole and systole, respectively. The HRD pulse sequence sampling scheme was 5(n)3/4(n)3(n)2, where n was determined by the heart rate to ensure adequate magnetization recovery. Image quality of T1 maps was assessed. T1 times were measured in myocardium and blood. Extracellular volume fraction (ECV) was calculated.
Results
In volunteers with repeated T1 mapping, the myocardial native T1 and ECV generated from the 1st fixed sampling scheme were smaller than from the 1st HRD and 2nd fixed sampling scheme. In healthy volunteers, the overall native T1 times and ECV of the left ventricle (LV) in diastolic T1 maps were greater than in systolic T1 maps (P < 0.01, P < 0.05). In the 3 AF patients that had received electrical cardioversion therapy, the myocardial native T1 times and ECV generated from the fixed sampling scheme were smaller than in the 1st and 2nd HRD sampling scheme (all P < 0.05). In patients with AF (HR: 88 ± 20 bpm, HR fluctuation: 12 ± 9 bpm), more T1 maps with artifact were found in diastole than in systole (P < 0.01). The overall native T1 times and ECV of the left ventricle (LV) in diastolic T1 maps were greater than systolic T1 maps, either with fixed or HRD sampling scheme (all P < 0.05).
Conclusion
Systolic MOLLI T1 mapping with heart-rate-dependent pulse sequence scheme can improve image quality and avoid T1 underestimation. It is feasible and with further validation may extend clinical applicability of T1 mapping to patients with atrial fibrillation.
doi:10.1186/s12968-016-0232-7
PMCID: PMC4793619  PMID: 26980571
T1 mapping; Modified Look-Locker inversion recovery; Extracellular volume fraction; Atrial fibrillation; Cardiovascular magnetic resonance
3.  Polymorphisms of the IL27 gene in a Chinese Han population complicated with pre-eclampsia 
Scientific Reports  2016;6:23029.
IL-27 could inhibit the development of Th17 cells, and the Th17/regulatory T-cell imbalance may reverse maternal tolerance in pre-eclampsia (PE). The aim of this study was to investigate the association between genetic polymorphisms in IL27 with PE. Three SNPs in IL27 (rs153109, rs17855750, and rs181206) were genotyped in a Chinese Han cohort of 1040 PE patients and 1247 normal pregnant women using the TaqMan allelic discrimination real-time PCR method. The CC genotypic distribution of rs153109 was significantly higher among cases than controls (19.1% versus 13.3%, odds ratio [OR]: 1.54, 95% confidence interval [CI]: 1.23–1.93, p < 0.001), and the CT genotype was found to be significantly lower in cases than controls (41.7% versus 49.0%, OR: 0.74, 95% CI: 0.63–0.88, p < 0.001), disputing existing reports indicating the allele frequency of rs153109 is not significantly different between PE patients and controls. Additionally, the CC genotype of rs153109 was significantly more prevalent in PE cases than controls using a recessive model (p < 0.001). The allelic and genotypic frequencies of rs17855750 and rs181206 were not significantly different between two groups. Our results reveal that IL27 polymorphisms may be involved in the development of PE in Chinese Han population.
doi:10.1038/srep23029
PMCID: PMC4789744  PMID: 26971578
4.  Elevated D-dimer and fibrinogen levels in serum of preoperative bone fracture patients 
SpringerPlus  2016;5:161.
The changes of coagulation parameters in preoperative fracture patients reflect the coagulation status before surgery. We did retrospective assessment of preoperative fracture patients (n = 113) admitted to the hospital between September 2013 and September 2014. The control group were selected from healthy adults (n = 113) with matched age and gender. Platelet, PT INR, APTT, fibrinogen (FIB) and D-dimer values were collected and analyzed. PT INR level was 1.043 ± 0.119, APTT was 31.91 ± 7.56 s, FIB was 320.6 ± 71.8 mg/dl and D-dimer was 1283 ± 1582 ng/ml for the fracture patients. For the control group, PT INR level was 0.9976 ± 0.0602, APTT was 33.22 ± 2.55 s, FIB was 277.3 ± 44.7 mg/dl and D-dimer was 97.53 ± 63.90 ng/ml. Meanwhile, D-dimer levels of different sites of fractures were also measured: Femora 2448 ± 1961 ng/ml; Humerus 792.4 ± 691.2 ng/ml; Ulna/Radius 619.4 ± 843.7 ng/ml; Vertebra 647.7 ± 787.1 ng/ml; Tibia/Fibula 496.3 ± 268.8 ng/ml; Clavicle 260.9 ± 170.9 ng/ml; Ankle 415.4 ± 286.6 ng/ml. To conclude, D-dimer and fibrinogen levels get higher in preoperative fracture patients than controls. Besides, D-dimer levels are significantly different among different locations of fractures, and our data revealed that D-dimer levels of Femora fracture were higher than other sites.
doi:10.1186/s40064-016-1817-1
PMCID: PMC4766159  PMID: 27026858
Fracture; D-dimer; Fibrinogen; PT INR; APTT
5.  Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts 
Scientific Reports  2016;6:20409.
Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface.
doi:10.1038/srep20409
PMCID: PMC4746586  PMID: 26857012
6.  Combating HER2-overexpressing breast cancer through induction of calreticulin exposure by Tras-Permut CrossMab 
Oncoimmunology  2015;4(3):e994391.
Although trastuzumab has succeeded in breast cancer treatment, acquired resistance is one of the prime obstacles for breast cancer therapies. There is an urgent need to develop novel HER2 antibodies against trastuzumab resistance. Here, we first rational designed avidity-imporved trastuzumab and pertuzumab variants, and explored the correlation between the binding avidity improvement and their antitumor activities. After characterization of a pertuzumab variant L56TY with potent antitumor activities, a bispecific immunoglobulin G-like CrossMab (Tras-Permut CrossMab) was generated from trastuzumab and binding avidity-improved pertuzumab variant L56TY. Although, the antitumor efficacy of trastuzumab was not enhanced by improving its binding avidity, binding avidity improvement could significantly increase the anti-proliferative and antibody-dependent cellular cytotoxicity (ADCC) activities of pertuzumab. Further studies showed that Tras-Permut CrossMab exhibited exceptional high efficiency to inhibit the progression of trastuzumab-resistant breast cancer. Notably, we found that calreticulin (CRT) exposure induced by Tras-Permut CrossMab was essential for induction of tumor-specific T cell immunity against tumor recurrence. These data indicated that simultaneous blockade of HER2 protein by Tras-Permut CrossMab could trigger CRT exposure and subsequently induce potent tumor-specific T cell immunity, suggesting it could be a promising therapeutic strategy against trastuzumab resistance.
doi:10.4161/2162402X.2014.994391
PMCID: PMC4404837  PMID: 25949918
calreticulin exposure; CrossMab; HER2-overexpressing breast cancer; pertuzumab; trastuzumab; T cell immunity; ADCC; antibody-dependent cellular cytotoxicity; CDR, complementarity determining region; CH1, constant heavy chain 1; CL, constant light chain; CRT, calreticulin; FCM, flow cytometry; HER, human epidermal growth factor receptor; HER2-ECD, extracellular domain of HER2; LDH, lactate dehydrogenase; mAb, monoclonal antibody; PBMCs, peripheral blood mononuclear cells; PI3K, phosphatidylinositol 3-kinase; SEC, size-exclusion chromatography
7.  Metabolic reprogramming in cancer cells: glycolysis, glutaminolysis, and Bcl-2 proteins as novel therapeutic targets for cancer 
Nearly a century ago, Otto Warburg made the ground-breaking observation that cancer cells, unlike normal cells, prefer a seemingly inefficient mechanism of glucose metabolism: aerobic glycolysis, a phenomenon now referred to as the Warburg effect. The finding that rapidly proliferating cancer cells favors incomplete metabolism of glucose, producing large amounts of lactate as opposed to synthesizing ATP to sustain cell growth, has confounded scientists for years. Further investigation into the metabolic phenotype of cancer has expanded our understanding of this puzzling conundrum, and has opened new avenues for the development of anti-cancer therapies. Enhanced glycolytic flux is now known to allow for increased synthesis of intermediates for sustaining anabolic pathways critical for cancer cell growth. Alongside the increase in glycolysis, cancer cells transform their mitochondria into synthesis machines supported by augmented glutaminolysis, supplying lipid production, amino acid synthesis, and the pentose phosphate pathways. Inhibition of several of the key enzymes involved in these pathways has been demonstrated to effectively obstruct cancer cell growth and multiplication, sensitizing them to apoptosis. The modulation of various regulatory proteins involved in metabolic processes is central to cancerous reprogramming of metabolism. The finding that members of one of the major protein families involved in cell death regulation also aberrantly regulated in cancers, the Bcl-2 family of proteins, are also critical mediators of metabolic pathways, provides strong evidence for the importance of the metabolic shift to cancer cell survival. Targeting the anti-apoptotic members of the Bcl-2 family of proteins is proving to be a successful way to selectively target cancer cells and induce apoptosis. Further understanding of how cancer cells modify metabolic regulation to increase channeling of substrates into biosynthesis will allow for the discovery of novel drug targets to treat cancer. In the present review, we focused on the recent developments in therapeutic targeting of different steps in glycolysis, glutaminolysis and on the metabolic regulatory role of Bcl-2 family proteins.
doi:10.1186/s12957-016-0769-9
PMCID: PMC4721116  PMID: 26791262
8.  EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway 
Journal of Virology  2015;89(13):6608-6618.
ABSTRACT
The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway.
IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.
doi:10.1128/JVI.00364-15
PMCID: PMC4468487  PMID: 25878102
9.  Insulin-like growth factor binding protein-3 is a new predictor of radiosensitivity on esophageal squamous cell carcinoma 
Scientific Reports  2015;5:17336.
Insulin-like growth factor binding protein-3 (IGFBP-3) plays an essential role in radiosensitivity of esophageal squamous cell carcinoma (ESCC). However, the underlying mechanism is not completely understood. Here, we observed that IGFBP-3 had favorable impact on the tumorigenicity of ESCC cells in nude mice by using an in vivo imaging system (IVIS) to monitor tumor growth treated with ionizing radiation (IR). Downregulation of IGFBP-3 expression enhanced tumor growth, inhibited anti-proliferative and apoptotic activity and result in IR resistance in vivo. Cell cycle antibody array suggested that silencing IGFBP-3 promoted transition from G0/G1 to S phase, perhaps though influencing Smad3 dephosphorylation and retinoblastoma protein (Rb) phosphorylation. Downregulation of P21 and P27, and upregulation of p-P27 (phospho-Thr187), cyclin-dependent kinase 2 (CDK2), and cyclin E1 might contribute to the G0/G1 to S phase transition promoted by IGFBP-3. Our results suggest that Smad3-P27/P21-cyclin E1/CDK2-phosphorylated retinoblastoma protein pathways might be involved in this IGFBP-3 mediated radiosensitivity transition in ESCC.
doi:10.1038/srep17336
PMCID: PMC4680797  PMID: 26670461
10.  TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality 
Scientific Reports  2015;5:18454.
Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations.
doi:10.1038/srep18454
PMCID: PMC4680874  PMID: 26669855
11.  Measurement Equivalence of the Empowerment Scale for White and Black Persons with Severe Mental Illness 
Objective
The current study examined the measurement equivalence on a measure of personal empowerment for African American and White consumers of mental health services.
Methods
Confirmatory Factor Analysis was used to assess measurement equivalences of the 28-item Empowerment Scale (Rogers, Chamberlin, Ellison & Crean, 1997), using data from 1,035 White and 301 African American persons with severe mental illness.
Results
Metric invariance of the Empowerment Scale was supported, in that the factor structure and loadings were equivalent across groups. Scalar invariance was violated on three items; however, the impact of these items on scale scores was quite small. Finally, subscales of empowerment tended to be more highly inter-correlated for African American than for White respondents.
Conclusions and Implications for Practice
Results generally support the use of Empowerment Scale for ethnic group comparisons. However, subtle differences in the psychometric properties of this measure suggest that African Americans and White individuals may conceptualize the construct of empowerment in different ways. Specifically, African American respondents had a lower threshold for endorsing some items on the self-esteem and powerlessness dimensions. Further, White respondents viewed the three dimensions of empowerment (self-esteem, powerlessness and activism) as more distinct, whereas these three traits were more strongly interrelated for African Americans.
doi:10.1037/prj0000069
PMCID: PMC4254373  PMID: 24884300
12.  miR-485-5p acts as a negative regulator in gastric cancer progression by targeting flotillin-1 
MicroRNAs (miRNAs) play important roles in cancer progression including gastric cancer. miR-485-5p is reported as a potential suppressor in breast cancer, but its expression, cellular function and clinic features in gastric cancer is not known. In our study, we found that miR-485-5p expression was down-regulated in gastric cancer cell lines. miR-485-5p could inhibit gastric cancer cell growth in vitro and in vivo. We also found that miR-485-5p suppressed gastric cancer cell metastasis and sphere formation. It was confirmed flotillin-1 (Flot1) as a direct target of miR-485-5p, and up-regulation of miR-485-5p could decrease expression of Flot1 in gastric cancer cells. Further investigation showed that ectopic expression of Flot1 partially reversed the inhibition effect of enforced miR-485-5p expression on the malignant phenotypes of gastric cancer cells. The low expression of miR-485-5p in gastric cancer tissues was related to advanced clinical features and poorer prognosis. Our study suggested that miR-485-5p could be a potential prognostic marker and functions as a tumor suppressor in human gastric cancer by post-transcriptionally targeting Flot1.
PMCID: PMC4697701  PMID: 26807169
Gastric cancer; miR-485-5p; Flot1
13.  Zinc mediated hepatic stellate cell collagen synthesis reduction through TGF-β signaling pathway inhibition 
This study is to investigate the effect and underlying mechanism of Zinc (Zn) on hepatic stellate cell collagen synthesis. The proliferation and collagen synthesis ability of LX-2 cells were detected after adding Zn. The collagen synthesis related proteins of MMP-13 and TIMP1 along with TGF-β signaling pathway related proteins were detected by Western blot. The role of TGF-β signaling pathway in collagen synthesis inhibition was identified by TGF-β RI siRNA silencing. Compared with control group, LX-2 cell proliferation ability was significantly inhibited at all Zn concentrations (50 μM, 100 μM and 200 μM). Zn at 50 μM did not affect the protein content of αSMA and type I collagen while 100 μM and 200 μM Zn could significantly inhibit αSMA expression. Compared with control group, gene expression and protein content of MMP-13 in 200 μM Zn group was significantly increased while no difference in gene expression and protein content of TIMP1 was found. TGF-β RI content in 200 μM Zn group was significantly decreased and the protein content of TGF-β RII was not affected. MMP-13 expression was significantly increased after TGF-β RI siRNA silencing. Further results showed that in LX-2 cells those TGF-β RI expression was inhibited, LX-2 cell proliferation ability and the expression of synthesis collagen related proteins of αSMA and type I collagen were greatly decreased. Zn could significantly inhibit the expression of αSMA and type I collagen by inhibiting TGF-β RI expression and promoting MMP-13 expression.
PMCID: PMC4723807  PMID: 26884962
Collagen synthesis; Zn; hepatic fibrosis; hepatic stellate cells
14.  Reduced metallothionein expression induced by Zinc deficiency results in apoptosis in hepatic stellate cell line LX-2 
The present study is to investigate the molecular mechanism by which Zinc (Zn) deficiency induces apoptosis in hepatic stellate cells. LX-2 cells were incubated with N,N,N’,N’-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN; 5 μM, 10 μM and 25 μM) for 24 h. MTT assay was used to test the proliferation ability of LX-2 cells. Flow cytometry was performed to detect cell apoptosis. Western blotting assay was employed to determine the expression of metallothionein (MT). Atomic absorption spectroscopy was performed to measure intracellular reactive oxygen species content. To test the activity of mitochondria, respiratory control rate was tested. To investigate the activation of apoptotic signaling pathway, cytochrome C oxidase activity was determined. TPEN effectively decreased the content of Zn in LX-2 cells. Zn deficiency led to the inhibition of proliferation and enhancement of apoptosis of LX-2 cells. Zn deficiency induced the inhibition of MT expression in LX-2 cells. Inhibition of MT expression induced by Zn deficiency resulted in enhanced reactive oxygen species content, impaired mitochondrial function and inhibition of cytochrome C oxidase activity. Intracellular MT content in LX-2 cells is reduced by Zn deficiency. Reduction in MT expression further increases intracellular ROS content, enhances oxidative stress, inhibits cytochrome C oxidase activity, impairs mitochondrial function, and finally leads to cell apoptosis.
PMCID: PMC4723824  PMID: 26884979
Zinc; metallothionein; apoptosis; hepatic stellate cells; LX-2 cells
15.  Expression profile of long non-coding RNAs in pancreatic cancer and their clinical significance as biomarkers 
Oncotarget  2015;6(34):35684-35698.
Long non-coding RNAs (lncRNAs) have shown great potential as powerful and non-invasive tumor markers. However, little is known about their value as biomarkers in pancreatic cancer (PC). We applied an Arraystar Human LncRNA Microarray which targeting 7419 lncRNAs to determine the lncRNA expression profile in PC and to screen the potential biomarkers. The most increased lncRNAs in PC tissues were HOTTIP-005, XLOC_006390, and RP11-567G11.1. Increased HOTTIP-005 and RP11-567G11.1 expression were poor prognostic factors for patients with PC (n = 144, p < 0.0001). The expression patterns of HOTTIP splice variants in PC were also detected. HOTTIP-005 and HOTTIP-001 were the first and second most increased HOTTIP splice variants, respectively. Plasma HDRF and RDRF (HOTTIP-005 and RP11-567G11.1 derived RNA fragments in plasma/serum) were present in stable form. Their levels were significantly increased in the patients with PC as compared to the healthy controls (n = 127 and 122 respectively, p < 0.0001) and the high levels were derived from PC. HDRF and RDRF levels are promising indicators for distinguishing patients with PC from those without PC. This study identified HOTTIP-005 and RP11-567G11.1 and their plasma fragments with the potential to be used as prognostic and diagnostic biomarkers of PC. Further large-scale prospective studies are needed to confirm our findings.
PMCID: PMC4742134  PMID: 26447755
long non-coding RNA; pancreatic cancer; splice variant; microarray; biomarker
16.  Profiles of responses of immunological factors to different subtypes of Kawasaki disease 
Background
The responses of immunological factors to different subtypes of Kawasaki disease (KD) remain poorly understood.
Methods
We recruited 388 patients with KD, 160 patients with infectious febrile disease and 85 normal children who served as control subjects. Both the levels and percentages of T lymphocyte subsets, natural killer cells (NK cells) and B cells were analyzed via flow cytometry. The levels of serum IgG, IgM, IgA and C3, C4 were assessed via velocity scatter turbidimetry.
Results
The most significant differences noted between the patients with infectious febrile disease and the normal children were the elevated levels of B cells, C3 and the ratio of CD4/CD8, and the decreased levels of CD8+ T cells and NK cells, as well as the moderate increase in the absolute value of the CD3+ cells. The decreased T cell levels and the elevated B cell levels were helpful in distinguishing typical KD from atypical KD; the elevated T cell levels, the elevated NK cell and B cell levels and the decreased B cell levels were helpful in predicting the effectiveness of IVIG; low C3 and C4 levels were linked with prodromal infections.
Conclusions
Lymphocytes subsets and complement markers may be useful in differentiating among the different subtypes of KD and in helping clinicians understand the pathophysiology of KD.
doi:10.1186/s12891-015-0744-6
PMCID: PMC4619387  PMID: 26497060
Kawasaki disease; Mucocutaneous lymph node syndrome; T lymphocyte subsets; Natural killer cells; B lymphocytes; Immunoglobulin; Coronary artery lesion
17.  A GntR family transcription factor positively regulates mycobacterial isoniazid resistance by controlling the expression of a putative permease 
BMC Microbiology  2015;15:214.
Background
Bacteria use transcriptional regulation to respond to environmental stresses. Specifically, exposure to antibacterial drugs is deemed to be an atypical stress, and altering transcriptional regulation in response to such stress can increase bacterial drug resistance. However, only a few transcription factors that regulate drug resistance have been reported.
Results
In the present study, a GntR family transcription factor, encoded by the MSMEG_0535 (Ms0535) gene, was shown to be an isoniazid (INH) resistance regulator in Mycobacterium smegmatis. When the Ms0535 gene was overexpressed, cells showed a significant increase in INH resistance. First, the interaction between Ms0535 and its own promoter was determined, and a conserved 26-bp palindromic DNA binding motif was identified using electrophoretic mobility shift and DNaseI footprinting assays. Second, quantitative reverse transcription-PCR assays showed that Ms0535 acted as a transcriptional activator, and positively regulated its own expression, as well as that of a permease encoded by the MSMEG_0534 (Ms0534) gene. Similar to the case for the Ms0535 gene, a recombinant Ms0534-overexpressing strain also exhibited increased INH resistance compared with the wild-type strain. Furthermore, we showed that Ms0535 and Ms0534 deletion strains were more sensitive to INH than the wild-type strain. Interestingly, overexpressing Ms0534 in the Ms0535 deletion strain enhanced its INH resistance. In contrast, the Ms0534 deletion strain was still sensitive to INH even when Ms0535 was overexpressed. These findings suggest that Ms0534 is an effector protein that affects INH resistance in M. smegmatis.
Conclusions
In summary, the GntR transcriptional regulator Ms0535 positively regulates INH resistance by transcriptionally regulating the expression of the Ms0534 permease in M. smegmatis. These results improve our understanding of the role of transcriptional regulation in INH drug resistance in mycobacteria.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0556-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-015-0556-8
PMCID: PMC4609117  PMID: 26474554
Mycobacteria; GntR; Isoniazid; Permease
18.  A new rodent model of cerebral hyperperfusion 
Background: Most studies of hyperperfusion and hyperperfusion syndrome after carotid endarterectomy or carotid stenting are based on clinical observation or meta-analyses in patients, whereas there is little corresponding fundamental research since proper animal model that can reproduce phenotype stably is not available. Therefore, we developed a rat model in which the pathophysiologic process of hyperperfusion can be mimicked. Methods: Global ischemia was induced by occluding bilateral common carotid arteries (BCAO) for 2 weeks. After that, the ligature was loosened to allow reperfusion. Phenylephrine was administered at concentrations of 10, 20, 30, 40, 50, 80, and 120 μg/mL for rapidly elevating blood pressure. Relative cerebral blood flow in relation to mean arterial pressure (MAP) was measured with Laser Doppler techniques. Sham animals underwent the same surgical operation but without artery-occlusion and received the same concentrations of phenylephrine. Results: Mild hypertension rapidly increased cerebral blood flow. Phenylephrine at different concentrations produced different effects on blood pressure. Hyperperfusion can be induced by phenylephrine at around 30 μg/mL, whereas phenylephrine at 80 μg/ml or higher induced arrhythmia and further cardiac dysfunction thus failed to induce hyperperfusion. Conclusions: Our data suggest that 30-50 μg/mL phenylephrine mildly elevated MAP and cerebral blood flow to the level exceeding 100% of baseline. This hyperperfusion model possesses several advantages including high phenotype reproducibility, low experimental failure rate and low animal mortality rate. It can be applied to study carotid stenosis or ischemia/reperfusion injury in rats.
PMCID: PMC4694234  PMID: 26770334
Bilateral carotid artery occlusion; hyperperfusion; hyperperfusion syndrome; carotid endarterectomy; carotid stenting; cerebral blood flow; phenylephrine
19.  Paradoxical effect of rapamycin on inflammatory stress-induced insulin resistance in vitro and in vivo 
Scientific Reports  2015;5:14959.
Insulin resistance is closely related to inflammatory stress and the mammalian target of rapamycin/S6 kinase (mTOR/S6K) pathway. The present study investigated whether rapamycin, a specific inhibitor of mTOR, ameliorates inflammatory stress-induced insulin resistance in vitro and in vivo. We used tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) stimulation in HepG2 hepatocytes, C2C12 myoblasts and 3T3-L1 adipocytes and casein injection in C57BL/6J mice to induce inflammatory stress. Our results showed that inflammatory stress impairs insulin signaling by reducing the expression of total IRS-1, p-IRS-1 (tyr632), and p-AKT (ser473); it also activates the mTOR/S6K signaling pathway both in vitro and in vivo. In vitro, rapamycin treatment reversed inflammatory cytokine-stimulated IRS-1 serine phosphorylation, increased insulin signaling to AKT and enhanced glucose utilization. In vivo, rapamycin treatment also ameliorated the impaired insulin signaling induced by inflammatory stress, but it induced pancreatic β-cell apoptosis, reduced pancreatic β-cell function and enhanced hepatic gluconeogenesis, thereby resulting in hyperglycemia and glucose intolerance in casein-injected mice. Our results indicate a paradoxical effect of rapamycin on insulin resistance between the in vitro and in vivo environments under inflammatory stress and provide additional insight into the clinical application of rapamycin.
doi:10.1038/srep14959
PMCID: PMC4598825  PMID: 26449763
20.  Identification and Functional Analysis of MicroRNAs in Mice following Focal Cerebral Ischemia Injury 
Numerous studies have demonstrated that genes, RNAs, and proteins are involved in the occurrence and development of stroke. In addition, previous studies concluded that microRNAs (miRNAs or miRs) are closely related to the pathological process of ischemic and hypoxic disease. Therefore, the aims of this study were to quantify the altered expression levels of miRNAs in the infarct region 6 h after middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in mice using a large-scale miRNAs microarray. Firstly, MCAO-induced cerebral ischemic injuries were investigated by observing the changes of neurological deficits, infarct volume and edema ratio. One hundred and eighteen differentially expressed miRNAs were identified in the infarct region of mice following the MCAOs compared with sham group (p < 0.05 was considered as significant). Among these 118 significantly expressed microRNAs, we found that 12 miRNAs were up-regulated with fold changes lager than two, and 18 miRNAs were down-regulated with fold changes less than 0.5 in the infarct region of mice following the 6 h MCAOs, compared with the sham group. Then, these 30 miRNAs with expression in fold change larger than two or less than 0.5 was predicted, and the functions of the target genes of 30 miRNAs were analyzed using a bioinformatics method. Finally, the miRNA-gene network was established and the functional miRNA-mRNA pairs were identified, which provided insight into the roles of the specific miRNAs that regulated specified genes in the ischemic injuries. The miRNAs identified in this study may represent effective therapeutic targets for stroke, and further study of the role of these targets may increase our understanding of the mechanisms underlying ischemic injuries.
doi:10.3390/ijms161024302
PMCID: PMC4632751  PMID: 26473853
middle cerebral artery occlusion; miRNA microarray; gene ontology; Kyoto encyclopedia of genes and genomes; network
21.  A generalized target theory and its applications 
Scientific Reports  2015;5:14568.
Different radiobiological models have been proposed to estimate the cell-killing effects, which are very important in radiotherapy and radiation risk assessment. However, most applied models have their own scopes of application. In this work, by generalizing the relationship between “hit” and “survival” in traditional target theory with Yager negation operator in Fuzzy mathematics, we propose a generalized target model of radiation-induced cell inactivation that takes into account both cellular repair effects and indirect effects of radiation. The simulation results of the model and the rethinking of “the number of targets in a cell” and “the number of hits per target” suggest that it is only necessary to investigate the generalized single-hit single-target (GSHST) in the present theoretical frame. Analysis shows that the GSHST model can be reduced to the linear quadratic model and multitarget model in the low-dose and high-dose regions, respectively. The fitting results show that the GSHST model agrees well with the usual experimental observations. In addition, the present model can be used to effectively predict cellular repair capacity, radiosensitivity, target size, especially the biologically effective dose for the treatment planning in clinical applications.
doi:10.1038/srep14568
PMCID: PMC4585963  PMID: 26411887
22.  Role of IL-1β, IL-6, IL-8 and IFN-γ in pathogenesis of central nervous system neuropsychiatric systemic lupus erythematous 
We discussed the role of IL-1β, IL-6, IL-8 and IFN-γ in the pathogenesis of central nervous system neuropsychiatric systemic lupus erythematous (CNS-NPSLE). Serum and cerebrospinal fluid samples were collected from CNS-NPSLE patients, non-CNS SLE patients, patients with intracranial infection and normal subjects. Levels of IL-1β, IL-6, IL-8 and IFN-γ in serum and cerebrospinal fluid were detected by ELISA, and the results were compared across the groups. All subjects received cerebral MRI. The risk threshold for each cytokine in CNS-NPSLE group was set as 2.5%. The positive rates of cytokines for different lesions in cerebral MRI findings in CNS-NPSLE group were compared. The correlations between cytokine levels and cerebral MRI findings were analyzed. All groups did not show significant differences in age and gender (F=1.34, P>0.05; x 2=2.05, P>0.05); The IL-1β, IL-6, IL-8 and IFN-γ levels of serum and cerebrospinal fluid in CNS-NPSLE group were obviously higher than those of the normal control (serum Z14=6.22, 6.04, 6.22, 5.70; cerebrospinal fluid Z14=6.38, 7.10, 6.97, 6.34, P<0.0083); IL-1β, IL-6 and IL-8 levels of cerebrospinal fluid of CNS-NPSLE group were higher than those of the non-CNS SLE group (Z12=2.73, Z12=3.18, Z12=3.86; P<0.0083); IL-1β, IL-6, IL-8 and IFN-γ levels of cerebrospinal fluid of CNS-NPSLE group were higher than those of the serum (Z=3.19, 6.30, 5.44, 3.19, P<0.05); IL-6>20.0679 pg/ml and IL-8>87.1811 pg/ml in the cerebrospinal fluid predicted a higher risk of CNS-NPSLE (x 2=11.98, P<0.05; x 2=4.65, P<0.05); The positive rates of IL-1β and IL-6 in the cerebrospinal fluid of CNS-NPSLE patients with demyelinating diseases were considerably higher than those of CNS-NPSLE patients with normal MRI findings (x 2=10.89, P<0.005; x 2=18.47, P<0.005). The positive rates of IL-6 and IFN-γ in the cerebrospinal fluid of CNS-NPSLE patients presenting with multiple ischemic foci were significantly higher than those with normal MRI findings (x 2=5.56, P<0.005; x 2=14.59, P<0.005). Some cytokines are involved in the pathogenesis of CNS-NPSLE and correlated with cerebral MRI findings in CNS-NPSLE.
PMCID: PMC4659087  PMID: 26629199
Neuropsychiatric systemic lupus erythematous (NPSLE); central nervous system (CNS); cytokines; logistic regression analysis
23.  InbR, a TetR family regulator, binds with isoniazid and influences multidrug resistance in Mycobacterium bovis BCG 
Scientific Reports  2015;5:13969.
Isoniazid (INH), an anti-tuberculosis (TB) drug, has been widely used for nearly 60 years. However, the pathway through which Mycobacterium tuberculosis responds INH remain largely unclear. In this study, we characterized a novel transcriptional factor, InbR, which is encoded by Rv0275c and belongs to the TetR family, that is directly responsive to INH. Disrupting inbR made mycobacteria more sensitive to INH, whereas overexpressing inbR decreased bacterial susceptibility to the drug. InbR could bind specifically to the upstream region of its own operon at two inverted repeats and act as an auto-repressor. Furthermore, InbR directly bind with INH, and the binding reduced InbR’s DNA-binding ability. Interestingly, susceptibilities were also changed by InbR for other anti-TB drugs, such as rifampin, implying that InbR may play a role in multi-drug resistance. Additionally, microarray analyses revealed a portion genes of the inbR regulon have similar expression patterns in inbR-overexpressing strain and INH-treated wild type strain, suggesting that these genes, for example iniBAC, may be responsible to the drug resistance of inbR-overexpressing strain. The regulation of these genes by InbR were further assessed by ChIP-seq assay. InbR may regulate multiple drug resistance of mycobacteria through the regulation of these genes.
doi:10.1038/srep13969
PMCID: PMC4564863  PMID: 26353937
24.  ICSI treatment of severe male infertility can achieve prospective embryo quality compared with IVF of fertile donor sperm on sibling oocytes 
Asian Journal of Andrology  2015;17(5):845-849.
Azoospermia, cryptozoospermia and necrospermia can markedly decrease the ability of males to achieve pregnancy in fertile females. However, patients with these severe conditions still have the option to be treated by intracytoplasmic sperm injection (ICSI) to become biological fathers. This study analyzed the fertilization ability and the developmental viabilities of the derived embryos after ICSI treatment of the sperm from these patients compared with in vitro fertilization (IVF) treatment of the proven-fertile donor sperm on sibling oocytes as a control. On the day of oocyte retrieval, the number of sperm suitable for ICSI collected from two ejaculates or testicular sperm extraction was lower than the oocytes, and therefore, excess sibling oocytes were treated by IVF with donor sperm. From 72 couples (73 cycles), 1117 metaphase II oocytes were divided into 512 for ICSI and 605 for IVF. Compared with the control, husbands’ sperm produced a lower fertilization rate in nonobstructive azoospermia (65.4% vs 83.2%; P < 0.001), crytozoospermia (68.8% vs 75.5%; P < 0.05) and necrospermia (65.0% vs 85.2%; P < 0.05). The zygotes derived in nonobstructive azoospermia had a lower cleavage rate (96.4% vs 99.4%; P < 0.05), but the rate of resultant good-quality embryos was not different. Analysis of the rates of cleaved and good-quality embryos in crytozoospermia and necrospermia did not exhibit a significant difference from the control. In conclusion, although the sperm from severe male infertility reduced the fertilization ability, the derived embryos had potential developmental viabilities that might be predictive for the expected clinical outcomes.
doi:10.4103/1008-682X.146971
PMCID: PMC4577602  PMID: 25652630
azoospermia; cryptozoospermia; embryonic development; fertilization; infertility; necrospermia; sibling oocytes
25.  Micro-RNA-155 is induced by K-Ras oncogenic signal and promotes ROS stress in pancreatic cancer 
Oncotarget  2015;6(25):21148-21158.
The oncogenic K-Ras can transform various mammalian cells and plays a critical role in development of pancreatic cancer. MicroRNAs (miRNA) have been shown to contribute to tumorigenic progression. However, the nature of miRNAs involved in K-Ras transformation remains to be investigated. Here, by using microarray we identified miR-155 as the most upregulated miRNA after both acute and prolonged activation of K-Ras in a doxycyline-inducible system. Pharmacological inhibition of MAPK and NF-κB pathway blocked the induction of miR-155 in response to K-Ras activation. Overexpression of miR-155 caused inhibition of Foxo3a, leading to decrease of major antioxidants including SOD2 and catalase, and enhanced pancreatic cell proliferation induced by ROS generation. Importantly, correlations of K-Ras, miR-155 and Foxo3a were also validated in human pancreatic cancer tissues. Therefore, we propose that miR-155 plays an important role in oncogenic K-Ras transformation mediated by cellular redox regulation.
PMCID: PMC4673256  PMID: 26020803
K-Ras; miR-155; reactive oxygen species; pancreatic cancer

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