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1.  Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus–related hepatocellular carcinoma 
Nature genetics  2012;45(1):72-75.
To identify genetic susceptibility loci for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) in the Chinese population, we carried out a genome-wide association study (GWAS) in 2,514 chronic HBV carriers (1,161 HCC cases and 1,353 controls) followed by a 2-stage validation among 6 independent populations of chronic HBV carriers (4,319 cases and 4,966 controls). The joint analyses showed that HCC risk was significantly associated with two independent loci: rs7574865 at STAT4, Pmeta = 2.48 × 10−10, odds ratio (OR) = 1.21; and rs9275319 at HLA-DQ, Pmeta = 2.72 × 10−17, OR = 1.49. The risk allele G at rs7574865 was significantly associated with lower mRNA levels of STAT4 in both the HCC tissues and nontumor tissues of 155 individuals with HBV-related HCC (Ptrend = 0.0008 and 0.0002, respectively). We also found significantly lower mRNA expression of STAT4 in HCC tumor tissues compared with paired adjacent nontumor tissues (P = 2.33 × 10−14).
PMCID: PMC4105840  PMID: 23242368
2.  Angiopoietin-Like 4 Confers Resistance to Hypoxia/Serum Deprivation-Induced Apoptosis through PI3K/Akt and ERK1/2 Signaling Pathways in Mesenchymal Stem Cells 
PLoS ONE  2014;9(1):e85808.
Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.
PMCID: PMC3897528  PMID: 24465718
3.  Wnt1 Inhibits Hydrogen Peroxide-Induced Apoptosis in Mouse Cardiac Stem Cells 
PLoS ONE  2013;8(3):e58883.
Because of their regenerative and paracrine abilities, cardiac stem cells (CSCs) are the most appropriate, optimal and promising candidates for the development of cardiac regenerative medicine strategies. However, native and exogenous CSCs in ischemic hearts are exposed to various pro-apoptotic or cytotoxic factors preventing their regenerative and paracrine abilities.
Methods and Results
We examined the effects of H2O2 on mouse CSCs (mCSCs), and observed that hydrogen peroxide (H2O2) treatment induces mCSCs apoptosis via the caspase 3 pathway, in a dose-dependent manner. We then examined the effects of Wnt1 over-expression on H2O2-induced apoptosis in mCSCs and observed that Wnt1 significantly decreased H2O2-induced apoptosis in mCSCs. On the other hand, inhibition of the canonical Wnt pathway by the secreted frizzled related protein 2 (SFRP2) or knockdown of β-catenin in mCSCs reduced cells resistance to H2O2-induced apoptosis, suggesting that Wnt1 predominantly prevents H2O2-induced apoptosis through the canonical Wnt pathway.
Our results provide the first evidences that Wnt1 plays an important role in CSCs’ defenses against H2O2-induced apoptosis through the canonical Wnt1/GSK3β/β-catenin signaling pathway.
PMCID: PMC3606408  PMID: 23533594
4.  A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP 
Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.
PMCID: PMC4142787  PMID: 25105973
5.  Overexpression of Testes-Specific Protease 50 (TSP50) Predicts Poor Prognosis in Patients with Gastric Cancer 
Purpose. To investigate the expression of TSP50 protein in human gastric cancers and its correlation with clinical/prognostic significance. Methods. Immunohistochemistry (IHC) analysis of TSP50 was performed on a tissue microarray (TMA) containing 334 primary gastric cancers. Western blot was carried out to confirm the expression of TSP50 in gastric cancers. Results. IHC analysis revealed high expression of TSP50 in 57.2% human gastric cancer samples (191 out of 334). However, it was poorly expressed in all of the 20 adjacent nontumor tissues. This was confirmed by western blot, which showed significantly higher levels of TSP50 expression in gastric cancer tissues than adjacent nontumor tissues. A significant association was found between high levels of TSP50 and clinicopathological characteristics including junior age at surgery (P = 0.001), later TNM stage (P = 0.000), and present lymph node metastases (P = 0.003). The survival of gastric cancer patients with high expression of TSP50 was significantly shorter than that of the patients with low levels of TSP50 (P = 0.021). Multivariate Cox regression analysis indicated that TSP50 overexpression was an independent prognostic factor for gastric cancer patients (P = 0.017). Conclusions. Our data demonstrate that elevated TSP50 protein expression could be a potential predictor of poor prognosis in gastric cancer patients.
PMCID: PMC3985325  PMID: 24799889
6.  Novel Mechanism of Inhibition of Dendritic Cells Maturation by Mesenchymal Stem Cells via Interleukin-10 and the JAK1/STAT3 Signaling Pathway 
PLoS ONE  2013;8(1):e55487.
Mesenchymal stem cells (MSCs) can suppress dendritic cells (DCs) maturation and function, mediated by soluble factors, such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), and nitric oxide (NO). Interleukin-10 (IL-10) is a common immunosuppressive cytokine, and the downstream signaling of the JAK-STAT pathway has been shown to be involved with DCs differentiation and maturation in the context of cancer. Whether IL-10 and/or the JAK-STAT pathway play a role in the inhibitory effect of MSCs on DCs maturation remains controversial. In our study, we cultured MSCs and DCs derived from rat bone marrow under different culturing conditions. Using Transwell plates, we detected by ELISA that the level of IL-10 significantly increased in the supernatants of MSC-DC co-cultures at 48 hours. The cell immunofluorescence assay suggested that the MSCs secreted more IL-10 than the DCs in the co-cultures. Adding exogenous IL-10 to the DCs monoculture or MSC-DC co-cultures stimulated IL-10 and led to a decrease in IL-12, and lower expression of the DCs surface markers CD80, CD86, OX62, MHC-II and CD11b/c. Supplementing the culture with an IL-10 neutralizing antibody (IL-10NA) showed precisely the opposite effect of adding IL-10. Moreover, we demonstrated that the JAK-STAT signaling pathway is involved in inhibiting DCs maturation. Both JAK1 and STAT3 expression and IL-10 secretion decreased markedly after adding a JAK inhibitor (AG490) to the co-culture plate. We propose that there is an IL-10 positive feedback loop, which may explain our observations of elevated IL-10 and enhanced JAK1 and STAT3 expression. Overall, we demonstrated that MSCs inhibit the maturation of DCs through the stimulation of IL-10 secretion, and by activating the JAK1 and STAT3 signaling pathway.
PMCID: PMC3559548  PMID: 23383203
7.  An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer 
PLoS ONE  2012;7(7):e42138.
NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1+ A549 cells and H596-NQO1 cells, but not in NQO1− H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC.
PMCID: PMC3407158  PMID: 22848731
8.  Spontaneous retroperitoneal hemorrhage after hemodialysis involving anticoagulant agents 
In this paper, we described the symptoms and treatment of a patient with diabetic nephropathy accompanied by spontaneous retroperitoneal hemorrhage after hemodialysis. An elderly female patient with diabetic nephropathy presented with severe pain, numbness, and an increasing swelling in the left hip and left thigh after six sessions of hemodialysis involving the use of an antiplatelet drug and an anticoagulant agent. Her hemoglobin decreased to 46 g/L. An abdominal ultrasound showed a hematoma in the left retroperitoneal space, and computed tomography (CT) findings revealed a 6 cm×8 cm×10 cm hematoma in the left psoas muscle. After aggressive supportive therapy [the administration of packed red blood cell transfusion, carbazochrome sodium sulfonate injection, and continuous venovenous hemofiltration (CVVH)], the patient’s vital signs stabilized and her hemoglobin increased to 86 g/L. Repeat CT showed that the hematoma had been partially absorbed after two weeks. Eventually, the patient was discharged with stable vital signs. Physicians should be aware of the possibility of spontaneous retroperitoneal hemorrhage, particularly in patients with diabetic nephropathy undergoing hemodialysis involving the use of anticoagulant agents.
PMCID: PMC3348233  PMID: 22556180
Spontaneous retroperitoneal hemorrhage; Diabetic nephropathy; Hemodialysis; Nadroparin calcium
10.  Construction of cytogenetic map of Gossypium herbaceum chromosome 1 and its integration with genetic maps 
Cytogenetic map can provide not only information of the genome structure, but also can build a solid foundation for genetic research. With the developments of molecular and cytogenetic studies in cotton (Gossypium), the construction of cytogenetic map is becoming more and more imperative.
A cytogenetic map of chromosome 1 (A101) of Gossypium herbaceum (A1) which includes 10 bacterial artificial chromosome (BAC) clones was constructed by using fluorescent in situ hybridization (FISH). Meanwhile, comparison and analysis were made for the cytogenetic map of chromosome 1 (A101) of G. herbaceum with four genetic linkage maps of chromosome 1 (Ah01) of G. hirsutum ((AD)1) and one genetic linkage map of chromosome 1 of (A101) G. arboreum (A2). The 10 BAC clones were also used to be localized on G. raimondii (D5) chromosome 1 (D501), and 2 of them showed clear unique hybridized signals. Furthermore, these 2 BAC clones were also shown localized on chromosome 1 of both A sub-genome and D sub-genome of G. hirsutum.
The comparison of the cytogenetic map with genetic linkage maps showed that most of the identified marker-tagged BAC clones appearing same orders in different maps except three markers showing different positions, which might indicate chromosomal segmental rearrangements. The positions of the 2 BAC clones which were localized on Ah01 and Dh01 chromosomes were almost the same as that on A101 and D501 chromosomes. The corresponding anchored SSR markers of these 2 BAC clones were firstly found to be localized on chromosome D501 (Dh01) as they were not seen mapped like this in any genetic map reported.
PMCID: PMC4307992  PMID: 25628758
Cotton; BAC-FISH; Cytogenetic map
11.  Undifferentiated embryonal sarcoma of liver: a multi-institutional experience with 9 cases 
To summarize the clinicopathological features, therapeutic regimens and outcomes for the patients with undifferentiated embryonal sarcoma of the liver (UESL), 9 cases of UESL were retrospectively reviewed. Complete clinical history, lab studies, imaging examinations and pathological findings were collected for analysis. Overall survival and progression free survival were assessed by Kaplan-Meier Survival Analysis. The patients were 6 to 37 years old, and included 6 males and 3 females. The tumor size ranged from 5 to 26 cm. Pathologically, the tumors consisted of proliferations of medium sized spindle, oval or stellate shaped pleomorphic cells loosely or compactly arranged in an edematous or myxoid matrix, with scattered bizarre multinucleated giant cells. All of the 9 cases were treated with radical resections, 6 of 9 cases received chemothrepy for postoperative treatment. All follow-up data were available, 4 of 9 cases had recurrence, and 2 patients were died. Time to recurrence in these cases was 19, 4, 29, 14 months. The mean overall survival (OS) was 58.25 ± 9.1 months and the mean progression-free survival (PFS) was 39.55 ± 11.6 months. UESL is a potential treatable malignance when treated with combined multiagent chemotherapy after resection.
PMCID: PMC4313983
Liver; undifferentiated embryonal sarcoma; hepatectomy; prognosis
12.  mir-155 regulates cardiac allograft rejection by targing the expression of suppressor of cytokine signaling-1 (DOCS1) in dendritic cells 
Previously, we observed that mir-155 is induced during dendritic cell (DC) differentiation. We now demon-strated convincing evidence indicating that mir-155 promotes DC maturation and regulates its capacity for antigen presentation and induction of alloreactive T cell activation. Interestingly, the induction of miR-155 expression in DCs is dependent on the TLR4/Myd88/NF-κB signaling. Our mechanistic studies further revealed that SOCS1 is a direct target for mir-155, and by binding to its 3’UTR, mir-155 is likely to affect SOCS1 translation. Suppression of mir-155 expression in DCs significantly attenuated LPS-induced DC maturation along with reduced capability to stimulate allogeneic T cell proliferation. As a result, administration of antagomiR-155 provided protection for cardiac allografts from rejection. Together, our data support that suppression of miR-155 in DCs could be a viable therapeutic strategy for prevention and treatment of allograft rejection in clinical setting of transplantation.
PMCID: PMC4276246  PMID: 25550988
miR-155; dendritic cells; immune response; allograft
13.  OCT4 Expression and Vasculogenic Mimicry Formation Positively Correlate with Poor Prognosis in Human Breast Cancer 
To evaluate the prognostic value of OCT4 expression and vasculogenic mimicry (VM) in human breast cancer, we examined OCT4 expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-schiff) double staining on 90 breast cancer specimens. All patients were followed up for five–149 months following surgery. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between OCT4 expression and VM formation (p < 0.05). Both OCT4 expression and VM were also positively correlated with lymph node metastasis, higher histological grade, and Nottingham prognostic index (p < 0.05). Patients with OCT4 expression or VM formation exhibited poorer overall survival (OS) and disease-free survival (DFS) than OCT4-negative or VM-negative patients (p < 0.05). OCT4-positive/VM-positive patients also had the worst OS and DFS (p < 0.05). In multivariate survival analysis, VM, Nottingham prognostic index (NPI), and Her2 were independent prognostic factors related to OS and OCT4-positive/VM-positive patients, whereas NPI and Her2 were independent predictors of DFS. These results suggest that a combined OCT4 expression/VM could improve the prognostic judgment for breast cancer patients.
PMCID: PMC4264130  PMID: 25353179
OCT4; vasculogenic mimicry; breast cancer
14.  Involvement of inflammation-related miR-155 and miR-146a in diabetic nephropathy: implications for glomerular endothelial injury 
BMC Nephrology  2014;15:142.
MicroRNAs have been demonstrated to play an important role in the pathogenesis of diabetic nephropathy (DN). In this study, we investigated both the repertoire of miRNAs in the kidneys of patients with DN and their potential regulatory role in inflammation-mediated glomerular endothelial injury.
The miRNA expression profiling of the renal biopsy samples was performed by a microarray analysis; then, in situ hybridization and real-time polymerase chain reaction (PCR) were used to determine the localization and expression of two of the miRNAs significantly up-regulated in human DN kidney samples, miR-155 and miR-146a, in the kidney tissues from type 1 and type 2 DN rat models. Human renal glomerular endothelial cells (HRGECs) cultured under high-glucose conditions were transfected with miR-155 and miR-146a mimics, and the transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, and nuclear factor (NF)-κB expressions were examined by western blot, real-time PCR, and an electrophoresis mobility shift assay.
The expression of both miR-155 and miR-146a was increased more than fivefold in the kidney samples of the DN patients compared with the controls, and the miR-155 expression was closely correlated with the serum creatinine levels (R = 0.95, P = 0.004). During the induction and progression of the disease in type 1 and type 2 DN rat models, miR-155 and miR-146a were demonstrated to increase gradually. In vitro, high glucose induced the over-expression of miR-155 and miR-146a in the HRGECs, which, in turn, increased the TNF-α, TGF-β1, and NF-κB expression.
Taken together, these findings indicate that the increased expression of miR-155 and miR-146a in the DN patients and in the experimental DN animal models was found to contribute to inflammation-mediated glomerular endothelial injury.
PMCID: PMC4236663  PMID: 25182190
15.  Genome-wide association study in Chinese men identifies two new prostate cancer risk loci at 9q31.2 and 19q13.4 
Nature genetics  2012;44(11):1231-1235.
Prostate cancer risk–associated variants have been reported in populations of European descent, African-Americans and Japanese using genome-wide association studies (GWAS). To systematically investigate prostate cancer risk–associated variants in Chinese men, we performed the first GWAS in Han Chinese. In addition to confirming several associations reported in other ancestry groups, this study identified two new risk-associated loci for prostate cancer on chromosomes 9q31.2 (rs817826, P = 5.45 × 10−14) and 19q13.4 (rs103294, P = 5.34 × 10−16) in 4,484 prostate cancer cases and 8,934 controls. The rs103294 marker at 19q13.4 is in strong linkage equilibrium with a 6.7-kb germline deletion that removes the first six of seven exons in LILRA3, a gene regulating inflammatory response, and was significantly associated with the mRNA expression of LILRA3 in T cells (P < 1 × 10−4). These findings may advance the understanding of genetic susceptibility to prostate cancer.
PMCID: PMC4116636  PMID: 23023329
16.  MDRL lncRNA Regulates the Processing of miR-484 Primary Transcript by Targeting miR-361 
PLoS Genetics  2014;10(7):e1004467.
Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but whether lncRNAs operate in the processing of miRNA primary transcript is unclear. Also, whether lncRNAs are involved in the regulation of the mitochondrial network remains to be elucidated. Here, we report that a long noncoding RNA, named mitochondrial dynamic related lncRNA (MDRL), affects the processing of miR-484 primary transcript in nucleus and regulates the mitochondrial network by targeting miR-361 and miR-484. The results showed that miR-361 that predominantly located in nucleus can directly bind to primary transcript of miR-484 (pri-miR-484) and prevent its processing by Drosha into pre-miR-484. miR-361 is able to regulate mitochondrial fission and apoptosis by regulating miR-484 levels. In exploring the underlying molecular mechanism by which miR-361 is regulated, we identified MDRL and demonstrated that it could directly bind to miR-361 and downregulate its expression levels, which promotes the processing of pri-miR-484. MDRL inhibits mitochondrial fission and apoptosis by downregulating miR-361, which in turn relieves inhibition of miR-484 processing by miR-361. Our present study reveals a novel regulating model of mitochondrial fission program which is composed of MDRL, miR-361 and miR-484. Our work not only expands the function of the lncRNA pathway in gene regulation but also establishes a new mechanism for controlling miRNA expression.
Author Summary
Long non-coding RNAs (lncRNAs) have been shown to be involved in a wide range of biological functions. However, studies linking individual lncRNA to the mitochondrial fission program remain scarce. Also, it remains unknown whether lncRNAs can operate in the processing of miRNA primary transcript. Here, we provide causal evidence for the involvement of the lncRNA MDRL in the mitochondrial dynamics and the processing of miR-484 primary transcript in cardiomyocyte. We identified MDRL which can act as an endogenous ‘sponge’ that directly binds to miR-361 and downregulates its expression levels. miR-361 can directly bind to primary transcript of miR-484 and prevent its processing by Drosha into pre-miR-484. MDRL inhibits mitochondrial fission and apoptosis by miR-361 and miR-484. Our present study reveals a novel regulating model which is composed of MDRL, miR-361 and miR-484. Modulation of their levels may provide a new approach for tackling myocardial infarction.
PMCID: PMC4109843  PMID: 25057983
17.  Wayside Bearing Fault Diagnosis Based on a Data-Driven Doppler Effect Eliminator and Transient Model Analysis 
Sensors (Basel, Switzerland)  2014;14(5):8096-8125.
A fault diagnosis strategy based on the wayside acoustic monitoring technique is investigated for locomotive bearing fault diagnosis. Inspired by the transient modeling analysis method based on correlation filtering analysis, a so-called Parametric-Mother-Doppler-Wavelet (PMDW) is constructed with six parameters, including a center characteristic frequency and five kinematic model parameters. A Doppler effect eliminator containing a PMDW generator, a correlation filtering analysis module, and a signal resampler is invented to eliminate the Doppler effect embedded in the acoustic signal of the recorded bearing. Through the Doppler effect eliminator, the five kinematic model parameters can be identified based on the signal itself. Then, the signal resampler is applied to eliminate the Doppler effect using the identified parameters. With the ability to detect early bearing faults, the transient model analysis method is employed to detect localized bearing faults after the embedded Doppler effect is eliminated. The effectiveness of the proposed fault diagnosis strategy is verified via simulation studies and applications to diagnose locomotive roller bearing defects.
PMCID: PMC4062996  PMID: 24803197
fault diagnosis; locomotive bearing; wayside monitoring; Doppler effect; transient model
18.  Anthraquinone Derivatives as Potent Inhibitors of c-Met Kinase and the Extracellular Signaling Pathway 
ACS Medicinal Chemistry Letters  2013;4(4):408-413.
The aberrant function of c-Met kinase signaling pathway is ubiquitously involved in a broad spectrum of human cancers; thus, a strong rationale exists for targeting the kinase pathway in cancer therapy. Via integration of computational and experimental studies, anthraquinone derivatives were identified for the first time as potent c-Met kinase inhibitors in this research. The aberrant activation of the c-Met kinase pathway results from (TPR)-Met, MET gene mutation, or amplification and a hepatocyte growth factor (HGF)/scatter factor-dependent autocrine or paracrine mechanism. However, anthraquinone derivatives exclusively suppressed c-Met phosphorylation stimulated by HGF in A549 cells, indicating that the compounds possess the ability to block the extracellular HGF-dependent pathway. A surface plasmon resonance assay revealed that the most potent compound, 2a, shows a high binding affinity for HGF with an equilibrium dissociation constant of 1.95 μM. The dual roles of compound 2a demonstrate the potency of anthraquinone derivatives and provide a new design solution for the c-Met kinase signaling pathway.
PMCID: PMC4027229  PMID: 24900685
Anthraquinone derivatives; c-Met kinase inhibitors; binding affinity with HGF
19.  Cuticular differences associated with aridity acclimation in African malaria vectors carrying alternative arrangements of inversion 2La 
Parasites & Vectors  2014;7:176.
Principal malaria vectors in Africa, An. gambiae and An. coluzzii, share an inversion polymorphism on the left arm of chromosome 2 (2La/2L+a) that is distributed non-randomly in the environment. Genomic sequencing studies support the role of strong natural selection in maintaining steep clines in 2La inversion frequency along environmental gradients of aridity, and physiological studies have directly implicated 2La in heat and desiccation tolerance, but the precise genetic basis and the underlying behavioral and physiological mechanisms remain unknown. As the insect cuticle is the primary barrier to water loss, differences in cuticle thickness and/or epicuticular waterproofing associated with alternative 2La arrangements might help explain differences in desiccation resistance.
To test that hypothesis, two subcolonies of both An. gambiae and An. coluzzii were established that were fixed for alternative 2La arrangements (2La or 2L+a) on an otherwise homosequential and shared genetic background. Adult mosquitoes reared under controlled environmental conditions (benign or arid) for eight days post-eclosion were collected and analyzed. Measurements of cuticle thickness were made based on scanning electron microscopy, and cuticular hydrocarbon (CHC) composition was evaluated by gas chromatography–mass spectrometry.
After removing the allometric effects of body weight, differences in mean cuticle thickness were found between alternative 2La karyotypes, but not between alternative environments. Moreover, the thicker cuticle of the An. coluzzii 2La karyotype was contrary to the known higher rate of water loss of this karyotype relative to 2L+a. On the other hand, quantitative differences in individual CHCs and overall CHC profiles between alternative karyotypes and environmental conditions were consistent with expectation based on previous physiological studies.
Our results suggest that alternative arrangements of the 2La inversion are associated with differences in cuticle thickness and CHC composition, but that only CHC composition appears to be relevant for desiccation resistance. Differences in the CHC composition were consistent with previous findings of a lower rate of water loss for the 2L+a karyotype at eight days post-eclosion, suggesting that CHC composition is an important strategy for maintaining water balance in this genetic background, but not for 2La. Despite a higher rate of water loss at eight days, higher body water content of the 2La karyotype confers a level of desiccation resistance equivalent to that of the 2L+a karyotype.
PMCID: PMC3991895  PMID: 24721548
An. gambiae; An. coluzzii; Cuticular hydrocarbons; Chromosomal inversion; Cuticle; Desiccation resistance; GC-MS; M and S molecular forms
20.  Structural and biochemical insights into the V/I505T mutation found in the EIAV gp45 vaccine strain 
Retrovirology  2014;11:26.
The equine infectious anemia virus (EIAV) is a lentivirus of the Retrovirus family, which causes persistent infection in horses often characterized by recurrent episodes of high fever. It has a similar morphology and life cycle to the human immunodeficiency virus (HIV). Its transmembrane glycoprotein, gp45 (analogous to gp41 in HIV), mediates membrane fusion during the infection. However, the post-fusion conformation of EIAV gp45 has not yet been determined. EIAV is the first member of the lentiviruses for which an effective vaccine has been successfully developed. The attenuated vaccine strain, FDDV, has been produced from a pathogenic strain by a series of passages in donkey dermal cells. We have previously reported that a V/I505T mutation in gp45, in combination with other mutations in gp90, may potentially contribute to the success of the vaccine strain. To this end, we now report on our structural and biochemical studies of the gp45 protein from both wide type and vaccine strain, providing a valuable structural model for the advancement of the EIAV vaccine.
We resolved crystal structures of the ecto-domain of gp45 from both the wild-type EIAV and the vaccine strain FDDV. We found that the V/I505T mutation in gp45 was located in a highly conserved d position within the heptad repeat, which protruded into a 3-fold symmetry axis within the six-helix bundle. Our crystal structure analyses revealed a shift of a hydrophobic to hydrophilic interaction due to this specific mutation, and further biochemical and virological studies confirmed that the mutation reduced the overall stability of the six-helix bundle in post-fusion conformation. Moreover, we found that altering the temperatures drastically affected the viral infectivity.
Our high-resolution crystal structures of gp45 exhibited high conservation between the gp45/gp41 structures of lentiviruses. In addition, a hydrophobic to hydrophilic interaction change in the EIAV vaccine strain was found to modulate the stability and thermal-sensitivity of the overall gp45 structure. Our observations suggest that lowering the stability of the six-helix bundle (post-fusion), which may stabilizes the pre-fusion conformation, might be one of the reasons of acquired dominance for FDDV in viral attenuation.
PMCID: PMC3997929  PMID: 24656154
EIAV; gp45; Crystal structure; Stability; Vaccine strain; Heptad repeat; Pre-fusion conformation; Replication
21.  Ketamine-Induced Neuronal Damage and Altered N-Methyl-d-Aspartate Receptor Function in Rat Primary Forebrain Culture 
Toxicological Sciences  2012;131(2):548-557.
Ketamine, a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, is frequently used in pediatric general anesthesia. Accumulating evidence from animal experiments has demonstrated that ketamine causes neuronal cell death during the brain growth spurt. To elucidate the underlying mechanisms associated with ketamine-induced neuronal toxicity and search for approaches or agents to prevent ketamine’s adverse effects on the developing brain, a primary nerve cell culture system was utilized. Neurons harvested from the forebrain of newborn rats were maintained under normal control conditions or exposed to either ketamine (10µM) or ketamine plus l-carnitine (an antioxidant; 1–100µM) for 24h, followed by a 24-h withdrawal period. Ketamine exposure resulted in elevated NMDA receptor (NR1) expression, increased generation of reactive oxygen species (ROS) as indicated by higher levels of 8-oxoguanine production, and enhanced neuronal damage. Coadministration of l-carnitine significantly diminished ROS generation and provided near complete protection of neurons from ketamine-induced cell death. NMDA receptors regulate channels that are highly permeable to calcium, and calcium imaging data demonstrated that neurons exposed to ketamine had a significantly elevated amplitude of calcium influx and higher intracellular free calcium concentrations ([Ca2+]i) evoked by NMDA (50µM), compared with control neurons. These findings suggest that prolonged ketamine exposure produces an increase in NMDA receptor expression (compensatory upregulation), which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to elevated ROS generation and neuronal cell death. l-Carnitine appears to be a promising agent in preventing or reversing ketamine’s toxic effects on neurons at an early developmental stage.
PMCID: PMC3551423  PMID: 23065140
ketamine; NMDA receptor; reactive oxygen species; calcium influx; l-carnitine
22.  Suppression of KIF3B Expression Inhibits Human Hepatocellular Carcinoma Proliferation 
Digestive Diseases and Sciences  2013;59(4):795-806.
Human hepatocellular carcinoma (HCC) is one of the most common fatal cancers and an important health problem worldwide, but its mechanism is still unclear. Microtubule (MT) kinesin motor proteins orchestrate a variety of cellular processes (e.g. mitosis, motility and organelle transportation) and have been involved in human carcinogenesis. KIF3B, the kinesin superfamily of proteins (KIFs), plays an important role in the regulation of mitotic progression.
The expression of KIF3B and its involvement in HCC was investigated.
Western blot and immunohistochemistry were used to measure the expression of KIF3B protein in HCC and adjacent non-tumorous tissues in 57 patients and Cell Counting Kit-8 to analyze the effects of growth and interference of KIF3B in the cell cycle process.
KIF3B protein level was increased in HCC tissues compared with the adjacent non-tumorous tissues. It was significantly associated with histological differentiation, tumor size, the level of alpha fetal protein (AFP) and proliferation marker Ki-67. Over-expression of KIF3B was correlated with poor survival. Following release of HepG2 cells from serum starvation, the expression of KIF3B was up-regulated. Furthermore, suppression of KIF3B not only decreased cancer cell growth but also induced apoptosis of cells.
Our results suggested that KIF3B expression was upregulated in HCC tumor tissues and proliferating HCC cells, and an increased KIF3B expression was associated with poor overall survival. KIF3B over-expression is involved in the pathogenesis of hepatocellular carcinoma and may serve as a potential therapeutic target for human HCC.
PMCID: PMC3958719  PMID: 24368420
Human hepatocellular carcinoma (HCC); KIF3B; Cell proliferation; Pathogenesis
23.  UDP-Glucuronosyltransferase 1A Compromises Intracellular Accumulation and Anti-Cancer Effect of Tanshinone IIA in Human Colon Cancer Cells 
PLoS ONE  2013;8(11):e79172.
Background and Purpose
NAD(P)H: quinone oxidoreductase 1 (NQO1) mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation is the dominant metabolic pathway of tanshinone IIA (TSA), a promising anti-cancer agent. UGTs are positively expressed in various tumor tissues and play an important role in the metabolic elimination of TSA. This study aims to explore the role of UGT1A in determining the intracellular accumulation and the resultant apoptotic effect of TSA.
Experimental Approach
We examined TSA intracellular accumulation and glucuronidation in HT29 (UGT1A positive) and HCT116 (UGT1A negative) human colon cancer cell lines. We also examined TSA-mediated reactive oxygen species (ROS) production, cytotoxicity and apoptotic effect in HT29 and HCT116 cells to investigate whether UGT1A levels are directly associated with TSA anti-cancer effect. UGT1A siRNA or propofol, a UGT1A9 competitive inhibitor, was used to inhibit UGT1A expression or UGT1A9 activity.
Key Results
Multiple UGT1A isoforms are positively expressed in HT29 but not in HCT116 cells. Cellular S9 fractions prepared from HT29 cells exhibit strong glucuronidation activity towards TSA, which can be inhibited by propofol or UGT1A siRNA interference. TSA intracellular accumulation in HT29 cells is much lower than that in HCT116 cells, which correlates with high expression levels of UGT1A in HT29 cells. Consistently, TSA induces less intracellular ROS, cytotoxicity, and apoptotic effect in HT29 cells than those in HCT116 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol can decrease TSA glucuronidation and simultaneously improve its intracellular accumulation, as well as enhance TSA anti-cancer effect.
Conclusions and Implications
UGT1A can compromise TSA cytotoxicity via reducing its intracellular exposure and switching the NQO1-triggered redox cycle to metabolic elimination. Our study may shed a light in understanding the cellular pharmacokinetic and molecular mechanism by which UGTs determine the chemotherapy effects of drugs that are UGTs’ substrates.
PMCID: PMC3828323  PMID: 24244442
24.  Ultrasound measurement of the corpus callosum and neural development of premature infants 
Neural Regeneration Research  2013;8(26):2432-2440.
Length and thickness of 152 corpus callosa were measured in neonates within 24 hours of birth. Using ultrasonic diagnostic equipment with a neonatal brain-specific probe, corpus callosum length and thickness of the genu, body, and splenium were measured on the standard mid-sagittal plane, and the anteroposterior diameter of the genu was measured in the coronal plane. Results showed that corpus callosum length as well as thickness of the genu and splenium increased with tional age and birth weight, while other measures did not. These three factors on the standard mid-sagittal plane are therefore likely to be suitable for real-time evaluation of corpus callosum velopment in premature infants using cranial ultrasound. Further analysis revealed that thickness of the body and splenium and the anteroposterior diameter of the genu were greater in male infants than in female infants, suggesting that there are sex differences in corpus callosum size during the neonatal period. A second set of measurements were taken from 40 premature infants whose gestational age was 34 weeks or less. Corpus callosum measurements were corrected to a gestational age of 40 weeks, and infants were grouped for analysis depending on the outcome of a neonatal behavioral neurological assessment. Compared with infants with a normal neurological assessment, corpus callosum length and genu and splenium thicknesses were less in those with abnormalities, indicating that corpus callosum growth in premature infants is associated with neurobehavioral development during the early extrauterine stage.
PMCID: PMC4146107  PMID: 25206553
neural regeneration; neurogenesis; brain injury; premature infant; cranial ultrasound; neonate; corpus callosum; neonatal neurobehavior; grants-supported paper; neuroregeneration
25.  Regional gray matter atrophy and neuropsychologcal problems in relapsing-remitting multiple sclerosis 
Neural Regeneration Research  2013;8(21):1958-1965.
In multiple sclerosis, gray matter atrophy is extensive, and cognitive deficits and mood disorders are frequently encountered. It has been conjectured that focal atrophy is associated with emotional decline. However, conventional MRI has revealed that the pathological characteristics cannot fully account for the mood disorders. Moreover, there is no correlation between cognitive disorders and MRI results in clinically isolated syndromes or in cases of definite multiple sclerosis. In this case-control study, voxel-based morphometric analysis was performed on 11 subjects with relapsing-remitting multiple sclerosis, and the results show that these patients exhibit gray matter atrophy. Moreover, the gray matter atrophy in the superior and middle gyri of the right frontal lobe in patients with multiple sclerosis was correlated with scores from the Hamilton Anxiety Rating Scale. The scores obtained with the Repeatable Battery for the Assessment of Neuropsychological Status were associated with gray matter atrophy in the middle gyrus of the left frontal lobe, the superior and middle gyrus of the right frontal lobe, the middle gyrus of the left cingulate, the superior and middle gyri of the left frontal lobe, and the triangular area of the left frontal lobe. However, there was no statistical significance. These findings suggest that the cingulate and frontal cortices of the nant hemisphere are the most severely atrophic regions of the brain, and this atrophy is correlated with cognitive decline and emotional abnormalities.
PMCID: PMC4145907  PMID: 25206504
neural regeneration; neurodegeneration; MRI; relapsing-remitting multiple sclerosis; gray matter atrophy; cognitive; mood; voxel-based morphometry; neuroregeneration

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