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1.  The role, mechanism and potentially therapeutic application of microRNA-29 family in acute myeloid leukemia 
Cell Death and Differentiation  2013;21(1):100-112.
Abnormal proliferation, apoptosis repression and differentiation blockage of hematopoietic stem/progenitor cells have been characterized to be the main reasons leading to acute myeloid leukemia (AML). Previous studies showed that miR-29a and miR-29b could function as tumor suppressors in leukemogenesis. However, a comprehensive investigation of the function and mechanism of miR-29 family in AML development and their potentiality in AML therapy still need to be elucidated. Herein, we reported that the family members, miR-29a, -29b and -29c, were commonly downregulated in peripheral blood mononuclear cells and bone marrow (BM) CD34+ cells derived from AML patients as compared with the healthy donors. Overexpression of each miR-29 member in THP1 and NB4 cells markedly inhibited cell proliferation and promoted cell apoptosis. AKT2 and CCND2 mRNAs were demonstrated to be targets of the miR-29 members, and the role of miR-29 family was attributed to the decrease of Akt2 and CCND2, two key signaling molecules. Significantly increased Akt2, CCND2 and c-Myc levels in the AML cases were detected, which were correlated with the decreased miR-29 expression in AML blasts. Furthermore, a feed-back loop comprising of c-Myc, miR-29 family and Akt2 were found in myeloid leukemogenesis. Reintroduction of each miR-29 member partially corrected abnormal cell proliferation and apoptosis repression and myeloid differentiation arrest in AML BM blasts. An intravenous injection of miR-29a, -29b and -29c in the AML model mice relieved leukemic symptoms significantly. Taken together, our finding revealed a pivotal role of miR-29 family in AML development and rescue of miR-29 family expression in AML patients could provide a new therapeutic strategy.
doi:10.1038/cdd.2013.133
PMCID: PMC3857615  PMID: 24076586
acute myeloid leukemia; cell proliferation and apoptosis; miR-29 family; diagnostic markers; therapeutic targets
2.  Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8 
Cell Death and Differentiation  2013;21(1):59-68.
The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.
doi:10.1038/cdd.2013.73
PMCID: PMC3857625  PMID: 23787997
α factor; autophagy; apoptosis; BAX; endoplasmic reticulum stress; PERK
3.  Molecular mechanisms of ATP secretion during immunogenic cell death 
Cell Death and Differentiation  2013;21(1):79-91.
The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase- and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.
doi:10.1038/cdd.2013.75
PMCID: PMC3857631  PMID: 23852373
apoptosis; Beclin 1; caspases; endoplasmic reticulum stress; quinacrine; U2OS cells
4.  Sorafenib for advanced hepatocellular carcinoma (HCC): impact of rationing in the United Kingdom 
British Journal of Cancer  2013;109(4):888-890.
Background:
The prognosis for hepatocellular carcinoma (HCC) is dependent upon tumour stage, performance status (PS), severity of underlying liver disease, and the availability of appropriate therapies. The unavailability of sorafenib may have a significantly adverse effect on the prognosis of UK patients with advanced HCC. During the study period, access to sorafenib was at the discretion of local health funding bodies, a process that may delay or deny access to the drug and that remains in place for Wales, Scotland, and Northern Ireland. Here, we attempt to address the impact of this system on patients with advanced HCC in the United Kingdom.
Methods:
This is a retrospective study performed in the two largest specialist hepatobiliary oncology units in the United Kingdom. Funding applications were made to local funding bodies for patients with advanced HCC for whom sorafenib was considered appropriate (advanced HCC not suitable for loco-regional therapies, compensated chronic liver disease, PS 0–2).
Results:
A total of 133 applications were made, of which 57 (43%) were approved and 76 (57%) declined. Demographics and prognostic factors were balanced between the two groups. This cohort had a number of adverse prognostic features: patients were predominantly PS 1–2; the majority had multifocal disease with the largest lesion being >5 cm; and macroscopic vascular invasion, metastases, and AFP >1000 ng ml−1, were each present in one-third of cases. The median time from application to funding decision was 17 days (range 3–260 days). For the primary ‘intention-to-treat' analysis, median overall survival was 4.1 months when funding was declined, and 9.5 months when funding was approved (hazard ratio (HR) 0.48; 95% CI 0.3186–0.7267; P=0.0005).
Conclusion:
These data support the use of sorafenib for patients with advanced HCC as an effective intervention. In the United Kingdom, this applies to a relatively small group of patients, estimated to total ∼800 per year who, unfortunately, do not survive long enough to themselves lobby for the availability of this drug. These data provide a comparison of sorafenib with supportive care and demonstrate the potential detrimental impact on patient outcomes of rationing health-care resources on the basis of cost.
doi:10.1038/bjc.2013.410
PMCID: PMC3749577  PMID: 23880824
sorafenib; multikinase inhibitor; advanced hepatocellular cancer; primary care trusts; health-care rationing; supportive care
5.  Decreasing Excess Mortality of HIV-infected Patients Initiating Antiretroviral Therapy: Comparison with Mortality in General Population in China, 2003 – 2009 
Objective
To evaluate excess mortality across calendar time comparing HIV-infected patients receiving cART with the general Chinese population.
Methods
Patients receiving free cART through the National Free Antiretroviral Therapy Program (NFATP) between 1 January 2003 and 31 December 2009 were included. Observed mortality rates, excess mortality rates and standardized mortality ratios were calculated by calendar periods. Factors associated with excess mortality across calendar time were evaluated in multivariable Poisson regression models.
Results
Among 64,836 HIV-infected patients the observed and excess mortality rates in 2003/2004 were 9.5 deaths/100 person-years (95% confidence interval [95% CI]: 8.8, 10.2) and 9.1 (8.5, 9.8); in 2008/2009 these decreased to 5.6 (5.4, 5.8) and 5.2 (5.0, 5.4) respectively. The adjusted excess hazard ratio (eHR) for 2003/2004 in comparison to 2008/2009 was 1.27 (95% CI: 1.11, 1.45). Patients initiating cART at CD4 cell counts <50 cells/μL in comparison to ≥350 cells/μL had an adjusted eHR of 9.92 (95% CI: 8.59, 11.44). Patients starting cART at older ages also had greater excess mortality with an eHR of 1.63 (95% CI: 1.47, 1.82) comparing ages ≥ 45 to 18–29. Standardized mortality ratio results were consistent with those for excess mortality.
Conclusion
Substantial decreases in excess mortality were observed from 2003 to 2009 in China among HIV-infected patients receiving free cART. However, mortality among HIV-infected patients remained higher than the general Chinese population. As more efficacious first and second line cART regimens become increasingly available to Chinese HIV-infected patients, further reductions in overall and excess mortality are likely.
doi:10.1097/QAI.0b013e3182948d82
PMCID: PMC3752313  PMID: 23572014
HIV; Mortality; China; Antiretroviral Therapy
6.  Cav-1 deletion impaired hematopoietic stem cell function 
Bai, L | Shi, G | Zhang, L | Guan, F | Ma, Y | Li, Q | Cong, Y-S | Zhang, L
Cell Death & Disease  2014;5(3):e1140-.
A tightly controlled balance between hematopoietic stem and progenitor cell compartments is required to maintain normal blood cell homeostasis throughout life, and this balance is regulated by intrinsic and extrinsic cellular factors. Cav-1 is a 22-kDa protein that is located in plasma membrane invaginations and is implicated in regulating neural stem cell and embryonic stem cell proliferation. However, the role of Cav-1 in hematopoietic stem cell (HSC) function is largely unknown. In this study, we used Cav-1−/− mice to investigate the role of Cav-1 in HSCs function during aging. The results showed that Cav-1−/− mice displayed a decreased percentage of B cells and an increased percentage of M cells in the bone marrow and peripheral blood, and these changes were due to an increased number of HSCs. FACS analysis showed that the numbers of Lin−Sca1+c-kit+ cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs and multipotent progenitors were increased in Cav-1−/− mice compared with Cav-1+/+ mice, and this increase became more pronounced with aging. An in vitro clonogenic assay showed that LT-HSCs from Cav-1−/− mice had reduced ability to self-renew. Consistently, an in vivo competitive transplantation assay showed that Cav-1−/− mice failed to reconstitute hematopoiesis. Moreover, a Cav-1 deletion disrupted the quiescence of LSKs and promoted cell cycle progression through G2/M phase. In addition, we found that Cav-1 deletion impaired the ability of HSCs to differentiate into mature blood cells. Taken together, these data suggest that Cav-1-deficient cells impaired HSCs quiescence and induced environmental alterations, which limited HSCs self-renewal and function.
doi:10.1038/cddis.2014.105
PMCID: PMC3973224  PMID: 24675458
Cav-1; HSC; quiescence
7.  Magnetic Exchange Coupling and Anisotropy of 3d Transition Metal Nanowires on Graphyne 
Scientific Reports  2014;4:4014.
Applying two-dimensional monolayer materials in nanoelectronics and spintronics is hindered by a lack of ordered and separately distributed spin structures. We investigate the electronic and magnetic properties of one-dimensional zigzag and armchair 3d transition metal (TM) nanowires on graphyne (GY), using density functional theory plus Hubbard U (DFT + U). The 3d TM nanowires are formed on graphyne (GY) surfaces. TM atoms separately and regularly embed within GY, achieving long-range magnetic spin ordering. TM exchange coupling of the zigzag and armchair nanowires is mediated by sp-hybridized carbon, and results in long-range magnetic order and magnetic anisotropy. The magnetic coupling mechanism is explained by competition between through-bond and through-space interactions derived from superexchange. These results aid the realization of GY in spintronics.
doi:10.1038/srep04014
PMCID: PMC3918920  PMID: 24510164
8.  Tumor suppressor NDRG2 tips the balance of oncogenic TGF-β via EMT inhibition in colorectal cancer 
Shen, L | Qu, X | Ma, Y | Zheng, J | Chu, D | Liu, B | Li, X | Wang, M | Xu, C | Liu, N | Yao, L | Zhang, J
Oncogenesis  2014;3(2):e86-.
Transforming growth factor-beta (TGF-β), a pluripotent cytokine expressed in the colon, has a crucial but paradoxical role in colorectal cancer (CRC). TGF-β is a potent proliferation inhibitor of normal colon epithelial cells and acts as a tumor suppressor. However, TGF-β also promotes invasion and metastasis during late-stage CRC, thereby acting as an oncogene. Thus, understanding the factors behind the paradoxical roles of TGF-β and elucidating the mechanisms by which TGF-β-induced proliferation inhibition is impaired in CRC are necessary. Here, we found that the N-Myc tumor suppressor gene downstream-regulated gene NDRG2 (N-Myc downstream-regulated gene 2), which is a TGF-β-responsive gene, abrogated TGF-β-induced epithelial–mesenchymal transition (EMT) and further inhibited the invasion and migration of CRC cells. TGF-β positively induced NDRG2 expression through direct transactivation mediated by Sp1 and by abrogation of the repressive c-Myc/Miz-1 complex on NDRG2 promoter in normal epithelial cells. Aberrant hypermethylation of NDRG2, which could respond to TGF-β growth inhibition signaling, abrogated the inhibitory effect of NDRG2 in TGF-β-induced EMT in CRCs. Reduced NDRG2 expression was highly correlated with the invasion stage and metastasis of CRC. Our study establishes that NDRG2 is a new tumor suppressor gene that responds to TGF-β anti-proliferative signaling and tips the balance of oncogenic TGF-β during late-stage CRC.
doi:10.1038/oncsis.2013.48
PMCID: PMC3940918  PMID: 24492480
colorectal cancer; TGF-β; NDRG2; EMT; methylation
9.  Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro 
Wang, Q | Jiang, M | Wu, J | Ma, Y | Li, T | Chen, Q | Zhang, X | Xiang, L
Cell Death & Disease  2014;5(1):e1022-.
The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.
doi:10.1038/cddis.2013.539
PMCID: PMC4040706  PMID: 24457966
stress; RNASET2; melanocyte; apoptosis; TRAF2
10.  Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs 
Background
Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.
Methods
Hh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.
Results
siRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.
Conclusions
We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.
doi:10.1186/1756-8722-6-77
PMCID: PMC3852827  PMID: 24199791
NSCLC; Erlotinib resistance; Hh signaling; miRNAs; EMT; GDC-0449
11.  Study on Characteristics of Chemokine CXCL10 Gene Cloned from cDNA Expression Library of Ujumqin Sheep 
BioMed Research International  2013;2013:217942.
Chemokines were a major regulator of body's inflammatory and immune responses. In this study, the cDNA fragment of chemokine CXC ligand 10 (CXCL10) was cloned from the Ujumqin sheep ear marginal tissue cDNA expression library; the CXCL10 gene had 103 amino acids and a molecular weight of 11.47 kDa, and it shared a high homology among cattle, sheep, and goat, while a low homology compared with mouse. The CXCL10 protein had 4 conservative cysteine residues, located in 28, 30, 55, and 72 sites. The expression pattern and intracellular distribution of recombinant CXCL10 proteins in Ujumqin sheep fibroblast cells showed that there were green fluorescence signals both in cytoplasm and nucleolus after 24 h of transfection, the number of positive cells was increased with time, the peak level of fluorescence signal was reached after 48 h of transfection and the transfection efficiency was 33.3%; there was a significant decrease in fluorescence intensity after 72 h of transfection. Expression of recombinant CXCL10 gene in Escherichia coli had a time- and temperature-dependency on the amount of protein expression, and a small quantity of inducer was needed.
doi:10.1155/2013/217942
PMCID: PMC3804364  PMID: 24187661
12.  Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia☆ 
Placenta  2010;32(1):63-69.
Shedding of syncytiotrophoblast microparticles (MPs) from placenta to maternal blood occurs in normal pregnancy and is enhanced during preeclampsia (PE). The syncytiotrophoblast synthesizes plasminogen activator inhibitors (PAIs) which regulate fibrinolysis, as well as soluble forms of the fms-like tyrosine kinase (sFlt-1) and endoglin, which exert anti-angiogenic actions. An increase in the ratio of PAI-1/PAI-2 and elevated levels of sFlt-1 and sEng in maternal serum are linked to placental damage and maternal endothelial cell dysfunction in PE. The goal of the current study was to determine whether MPs released to maternal perfusate during dual perfusion contain these factors associated with placental pathophysiology in PE. Initially, high levels of alkaline phosphatase activity and Annexin V binding were found in MPs isolated by sequential centrifugation of maternal perfusates at 10,000 and 150,000×g(10 K and 150 K MPs), indicating their plasma membrane origin. ELISA revealed the presence of these factors at the following relative levels: Eng>PAI-2⋙ PAI-1>sFlt-1. Based on comparisons of their concentration in perfusates, MPs, and MP-free 150 K supernatants, we determined that MPs constitute a significant portion of Eng released by placenta. Flow cytometric analysis of 10 K MPs supported the levels of expression found by ELISA and indicated that Eng and PAI-2 were almost exclusively localized to the surface of MPs, a site with biological potential. These results indicate that MPs shed from the syncytial surface express factors which may alter the fibrinolytic and angiogenic balance at the maternal–fetal interface and play a role in the pathophysiology of PE.
doi:10.1016/j.placenta.2010.10.011
PMCID: PMC3762591  PMID: 21074265
Placental microparticles; Dual perfusion; Preeclampsia; Flt-1; Endoglin; Plasminogen activators
13.  Enhancer of zeste homolog 2 activates wnt signaling through downregulating CXXC finger protein 4 
Lu, H | Sun, J | Wang, F | Feng, L | Ma, Y | Shen, Q | Jiang, Z | Sun, X | Wang, X | Jin, H
Cell Death & Disease  2013;4(8):e776-.
Through silencing tumor suppressor genes, epigenetic changes can activate signaling pathways important to cancer development. In this report, we found an epigenetic contribution to the aberrant activation of wnt signaling in human gastric cancer. CXXC4 (CXXC finger protein 4) was identified as a novel target of EZH2 (enhancer of zeste homolog 2), and EZH2 promotes the activation of wnt singaling by downregulating CXXC4 expression. CXXC4 inhibits the growth of gastric cancer cells both in vitro and in vivo through inactivating wnt signaling. In contrast, depletion of CXXC4 activates wnt signaling and promotes the anchorage-independent growth of nontumor gastric epithelial cells. CXXC4 is downregulated in gastric carcinoma tissues and its downregulation is associated with poor outcome of gastric cancer patients (hazard ratio: 5.053, P<0.05). Through its binding to dishevelled (Dvl), CXXC4 stabilizes the destruction complex of β-catenin to inhibit wnt signaling. Two critical amino acid residues in CXXC4, K161 and T162 were found to be important to its binding to Dvl and the growth inhibitory effect of CXXC4. In summary, EZH2 promotes the activation of wnt signaling in gastric carcinogenesis through the downregulation of CXXC4 expression. CXXC4 is a novel potential tumor suppressor directly regulated by EZH2, and its expression is a significant prognosis factor for patients with early stages of gastric cancer.
doi:10.1038/cddis.2013.293
PMCID: PMC3763454  PMID: 23949225
EZH2; Wnt/β-catenin signaling; CXXC4; gastric cancer
14.  Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity 
Mi, Y-J | Hou, B | Liao, Q-M | Ma, Y | Luo, Q | Dai, Y-K | Ju, G | Jin, W-L
Cell Death and Differentiation  2012;19(7):1175-1186.
Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H2O2). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H2O2 treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure–function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290–562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.
doi:10.1038/cdd.2011.206
PMCID: PMC3374081  PMID: 22261619
Nogo-A; ROS; neuroprotection; Prdx2; cortical neurons
15.  Bright betatron X-ray radiation from a laser-driven-clustering gas target 
Scientific Reports  2013;3:1912.
Hard X-ray sources from femtosecond (fs) laser-produced plasmas, including the betatron X-rays from laser wakefield-accelerated electrons, have compact sizes, fs pulse duration and fs pump-probe capability, making it promising for wide use in material and biological sciences. Currently the main problem with such betatron X-ray sources is the limited average flux even with ultra-intense laser pulses. Here, we report ultra-bright betatron X-rays can be generated using a clustering gas jet target irradiated with a small size laser, where a ten-fold enhancement of the X-ray yield is achieved compared to the results obtained using a gas target. We suggest the increased X-ray photon is due to the existence of clusters in the gas, which results in increased total electron charge trapped for acceleration and larger wiggling amplitudes during the acceleration. This observation opens a route to produce high betatron average flux using small but high repetition rate laser facilities for applications.
doi:10.1038/srep01912
PMCID: PMC3665959  PMID: 23715033
16.  Push-Pull Effects of Three Plant Secondary Metabolites on Oviposition of the Potato Tuber Moth, Phthorimaea operculella 
The push-pull effects of three plant secondary metabolites, azadirachtin, eucalyptol, and heptanal, on the oviposition choices of potato tubers by the potato tuber moth, Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae) were tested in the laboratory. Azadirachtin at concentrations from 1.5 to 12 mg/L had a significant repellent effect on oviposition. Eucalyptol at concentrations from 3 to 12 mg/L promoted oviposition. Heptanal promoted oviposition at low concentrations from 0.1875 to 3.0 mg/L but repelled it at higher concentrations from 12 to 24 mg/L. The combination of azadirachtin (12 mg/L) with eucalyptol (3.0 mg/L) resulted in a significant pushpull effect of 56.3% on oviposition. The average maximum push-pull effects occurred with the combinations of azadirachtin with heptanal (12 and 0.375 mg/L, respectively; 38.7% push-pull effect), heptanal with eucalyptol (12 and 6 mg/L, respectively; 31.4% push-pull effect), and heptanal (high concentration) with heptanal (low concentration) (12.0 and 0.375 mg/L, respectively; 25% push-pull effect).
doi:10.1673/031.013.12801
PMCID: PMC4014036  PMID: 24786822
azadirachtin; eucalyptol; heptanal
17.  Circulating αKlotho influences phosphate handling by controlling FGF23 production 
The Journal of Clinical Investigation  2012;122(12):4710-4715.
The FGF23 coreceptor αKlotho (αKL) is expressed as a membrane-bound protein (mKL) that forms heteromeric complexes with FGF receptors (FGFRs) to initiate intracellular signaling. It also circulates as an endoproteolytic cleavage product of mKL (cKL). Previously, a patient with increased plasma cKL as the result of a translocation [t(9;13)] in the αKLOTHO (KL) gene presented with rickets and a complex endocrine profile, including paradoxically elevated plasma FGF23, despite hypophosphatemia. The goal of this study was to test whether cKL regulates phosphate handling through control of FGF23 expression. To increase cKL levels, mice were treated with an adeno-associated virus producing cKL. The treated groups exhibited dose-dependent hypophosphatemia and hypocalcemia, with markedly elevated FGF23 (38 to 456 fold). The animals also manifested fractures, reduced bone mineral content, expanded growth plates, and severe osteomalacia, with highly increased bone Fgf23 mRNA (>150 fold). cKL activity in vitro was specific for interactions with FGF23 and was FGFR dependent. These results demonstrate that cKL potently stimulates FGF23 production in vivo, which phenocopies the KL translocation patient and metabolic bone syndromes associated with elevated FGF23. These findings have important implications for the regulation of αKL and FGF23 in disorders of phosphate handling and biomineralization.
doi:10.1172/JCI64986
PMCID: PMC3533557  PMID: 23187128
18.  Super elastic strain limit in metallic glass films 
Scientific Reports  2012;2:852.
On monolithic Ni-Nb metallic glass films, we experimentally revealed 6.6% elastic strain limit by in-situ transmission electron microscopy observations. The origin of high elastic strain limit may link with high free volume in the film, causing the rearrangement of loosely bonded atomic clusters (or atoms) upon elastic deformation. This high elastic limit of metallic glass films will shed light on new application fields for metallic glasses, and also trigger more studies for deformation mechanism of amorphous materials in general.
doi:10.1038/srep00852
PMCID: PMC3496986  PMID: 23152943
19.  Expression of parafibromin in major renal cell tumors 
Parafibromin, encoded by HRPT2 gene, is a recently identified tumor suppressor. Complete and partial loss of its expression have been observed in hyperparathyroidism-jaw tumor (HPT-JT), parathyroid carcinoma, breast carcinoma, lung carcinoma, gastric and colorectal carcinoma. However, little has been known about its expression in renal tumors. In order to study the expression of parafibromin in a series of the 4 major renal cell tumors - clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), chromophobe renal cell carcinoma (chRCC) and oncocytoma, one hundred thirty nine renal tumors including 61 ccRCCs, 37 pRCCs, 22 chRCCs and 19 oncocytomas were retrieved and used for the construction of renal tissue microarrays (TMAs). The expression of parafibromin was detected by immunohistochemical method on the constructed TMAs. Positive parafibromin stains are seen in 4 out of 61 ccRCCs (7%), 7 out of 37 pRCCs (19%), 12 out of 23 chRCCs (52%) and all 19 oncocytomas (100%). Parafibromin expression varies significantly (P<8.8×10−16) among the four major renal cell tumors and were correlated closely with tumor types. No correlation of parafibromin expression with tumor staging in ccRCCs, pRCCs and chRCCs, and Fuhrman nuclear grading in ccRCCs and pRCCs was seen. In summary, parafibromin expression was strongly correlated with tumor types, which may suggest that it plays a role in the tumorigenesis in renal cell tumors.
doi:10.4081/ejh.2012.e39
PMCID: PMC3567758  PMID: 23361235
parafibromin; renal epithelial tumors.
20.  Apoptotic effects of hydrogen peroxide and vitamin C on chicken embryonic fibroblasts: redox state and programmed cell death 
Cytotechnology  2011;63(5):461-471.
The pro-apoptotic effects of hydrogen peroxide and the purported anti-apoptotic effects of Vitamin C on chicken embryonic fibroblasts were investigated. Hydrogen peroxide induced morphological changes in a dose dependent manner, and a myriad of autophagosomes were observed using transmission electron microscopy. Doxorubicin elicited alterations were not inhibited by co-incubation with Vitamin C except that mitochondrial structure was slightly improved. TUNEL assay, cytotoxicity analysis and flow cytometry revealed that the cytotoxicity, DNA fragmentation and apoptotic rates were dose dependent upon treatment with hydrogen peroxide. Calcium homeostasis was disrupted in a dose dependent manner, and cell cycle was blocked at G2/M checkpoint at low concentration and S/G2 checkpoint at high concentration respectively upon treatment with hydrogen peroxide. The administration of Vitamin C only has a modest effect against doxorubicin induced apoptosis, calcium homeostasis disruption and cell cycle arrest. This research demonstrated that the elevation of reactive oxygen species is sufficient to induce the apoptosis of chicken embryonic fibroblasts, whereas the administration of Vitamin C does not necessarily have certain anti-apoptotic effects, especially when the stimulus is not directly linked with redox state.
doi:10.1007/s10616-011-9360-y
PMCID: PMC3176935  PMID: 21822683
Chicken; Embryonic fibroblasts; Hydrogen peroxide; Vitamin C; Apoptosis; Redox state
21.  Network correlates of the cognitive response to levodopa in Parkinson disease 
Neurology  2011;77(9):858-865.
Objective:
Cognitive dysfunction is common in Parkinson disease (PD), even early in its clinical course. This disease manifestation has been associated with impaired verbal learning performance as well as abnormal expression of a specific PD-related cognitive spatial covariance pattern (PDCP). It is not known, however, how this metabolic network relates to the cognitive response to dopaminergic therapy on the individual patient level.
Methods:
We assessed treatment-mediated changes in verbal learning and PDCP expression in 17 patients with PD without dementia who underwent cognitive testing and metabolic imaging in the unmedicated and levodopa-treated conditions. We also determined whether analogous changes were present in 12 other patients with PD without dementia who were evaluated before and during the treatment of cognitive symptoms with placebo.
Results:
Levodopa-mediated changes in verbal learning correlated with concurrent changes in PDCP expression (r = −0.60, p < 0.01). The subset of patients with meaningful cognitive improvement on levodopa (n = 8) exhibited concurrent reductions in PDCP expression (p < 0.01) with treatment; network modulation was not evident in the remaining subjects. Notably, the levodopa cognitive response correlated with baseline PDCP levels (r = 0.70, p = 0.002). By contrast, placebo did not affect PDCP expression, even in the subjects (n = 7) with improved verbal learning during treatment.
Conclusions:
These findings suggest that cognitive dysfunction in PD may respond to treatment depending upon the degree of baseline PDCP expression. Quantification of treatment-mediated network changes can provide objective information concerning the efficacy of new agents directed at the cognitive manifestations of this disease.
doi:10.1212/WNL.0b013e31822c6224
PMCID: PMC3162641  PMID: 21849641
22.  Isolation and biological characterization of chicken amnion epithelial cells 
Amniotic epithelial cells (AECs) express Oct4, Nanog and Sox-2, which are necessary for maintaining the undifferentiated state of pluripotent stem cells. AECs additionally express CK19, which is a specific marker of epithelial cells, both in vivo and in vitro. In this research, we investigated the biological characteristics and potential for cell therapy of AECs from 6-day-old chicken embryos. We induced the AECs to differentiate into pancreatic islet-like cells (endoderm), adipocytes and osteoblasts (mesoderm) and neural-like cells (ectoderm), and used immunofluorescence and RT-PCR to detect the expression of AECs specific markers. To assess the differentiation capacity of AECs, passage 3 cells were induced to differentiate into adipocytes, osteoblasts, pancreatic islet-like cells and neural-like cells. The AEC markers, Oct-4, Nanog, Sox-2 and CK19, were all positively expressed. Cloning efficiency decreased with increasing passage number. Passage 3 AECs were successfully induced to differentiate into pancreatic islet-like cells, osteoblasts, adipocytes, and neural-like cells. These results suggested that AECs isolated from chicken embryos exhibited the characteristics of the multipotent stem cells. AECs may therefore be ideal candidates for cellular transplantation therapy and tissue engineering.
doi:10.4081/ejh.2012.e33
PMCID: PMC3493979  PMID: 23027349
chicken; AECs; biological research; differentiation capacity.
23.  Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma 
Blood Cancer Journal  2012;2(8):e82-.
Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK+ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK+ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK+ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2active cells, but not Sox2inactive cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK+ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.
doi:10.1038/bcj.2012.27
PMCID: PMC3432482  PMID: 22885405
Sox2; transcriptional activity; NPM-ALK; STAT3; tumorigenicity
24.  Changes in anesthesia related factors in ambulatory knee and shoulder surgery: United States 1996–2006 
Background
Analyses of existing nationally representative information on how changes in ambulatory orthopedic surgery have affected anesthesia practice over time are rare. We sought to characterize temporal changes in factors surrounding ambulatory orthopedic surgery and anesthesia.
Methods
Data from the National Survey of Ambulatory Surgery for the years of 1996 and 2006 were analyzed. Entries indicating the performance of knee ligamentoplasty, meniscectomy or shoulder arthroscopy were identified and included in the sample. Temporal changes in a number of variables associated with orthopedic ambulatory surgery were assessed, including: 1) the number of procedures being performed, 2) patient and health care system related demographics and 3) anesthesia related variables.
Results
Nationwide, the total number of ligamentoplasties, meniscectomies and shoulder arthroscopies increased from 1996 to 2006 by 66% (N=258,932 to N=428,712), 51% (N=456,698 to N=690,164), and 349% (N=93,105 to N=418,188) respectively (P<0.0001). Between 1996 and 2006 the use of peripheral nerve blocks increased from 0.6% to 9.8% for meniscectomies (P<0.0001), from 1.5% to 13.7% for ligamentoplasties (P<0.0001), and from 11.5% to 23.9% for shoulder arthroscopies (P<0.0001), respectively. Neuraxial anesthesia utilization fell from 11.8% to 6.3% for meniscectomies (P<0.0001) and 13.6% to 7.3% for ligamentoplasties (P<0.0001) from 1996 to 2006, respectively.
Conclusion
Substantial increases in the number of ambulatory knee and shoulder procedures occurred over time, relating to increased demand for anesthesia providers in this field. Trends towards increased use of peripheral nerve blocks may have to be considered by educators when preparing residents for practice.
doi:10.1097/AAP.0b013e318217703c
PMCID: PMC3121915  PMID: 21490521
25.  Smoking initiation is followed by the early acquisition of epigenetic change in cervical epithelium: a longitudinal study 
British Journal of Cancer  2011;104(9):1500-1504.
Background:
To prove a causal link between an epigenetic change and an environmental or behavioural risk factor for a given disease, it is first necessary to show that the onset of exposure precedes the first detection of that epigenetic change in subjects who are still free of disease.
Methods:
Towards this end, a cohort of women aged 15–19 years, recruited soon after they first had sexual intercourse, were used to provide sequential observations on the relationship between cigarette smoking and the detection in cervical cytological samples of methylated forms of CDKN2A (p16) using nested methylation-specific polymerase chain reaction.
Results:
Among women who remained cytologically normal and who tested negative for human papillomavirus DNA in cervical smears during follow-up, those who first started to smoke during follow-up had an increased risk of acquiring CDKN2A methylation compared with never-smokers (odds ratio=3.67; 95% confidence interval 1.09–12.33; P=0.04).
Conclusion:
Smoking initiation is associated with the appearance of methylated forms of CDKN2A.
doi:10.1038/bjc.2011.113
PMCID: PMC3101930  PMID: 21487403
smoking; epigenetic; methylation; cohort study; cervix

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