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1.  Platelet factor 4 protects bone marrow mesenchymal stem cells from acute radiation injury 
The British Journal of Radiology  2014;87(1040):20140184.
The aim of this study was to find a new radiation protector, platelet factor 4 (PF4) and to identify its effect on haemopoietic microenvironment in vitro and in vivo.
Radiation damage on bone marrow mesenchymal stem cells ex and in vitro was set up as models. Growth curve analysis, clonogenic survival assay, FACSCalibur™ (BD Immunocytometry Systems, San Jose, CA), 5-ethynyl-2′-deoxyuridine immunofluorescence staining and quantitative reverse transcription–polymerase chain reaction were employed to assess the characterization of bone marrow mesenchymal stem cells (BMSCs), proliferation, apoptosis, cell cycle and gene expression.
A dose- and time-dependent enhancement of cell viability and survival was observed for PF4 treatment along with 500 cGy γ-radiation in vitro. The same phenomena were noted in vivo, including enhancement of adherence and proliferation ability while inhibition of cell apoptosis, which were associated with a short-term decrease in the G0/G1 ratio owing to S phase arrest. These were accompanied with enhanced Bcl-2 expression and p53/p21 loss.
These results uncover that PF4 might be a novel therapeutic approach, which could reduce DNA damage and increase survival of BMSCs, in part, by inhibiting p53/p21 axis and facilitating DNA damage repair.
Advances in knowledge:
This study explores the feasibility of a new radioprotector and hence may be clinically important.
PMCID: PMC4112396  PMID: 24922360
2.  Long-term, regular remote ischemic preconditioning improves endothelial function in patients with coronary heart disease 
Remote ischemic preconditioning (RIPre) can prevent myocardial injury. The purpose of this study was to assess the beneficial effects of long-term regular RIPre on human arteries. Forty patients scheduled for coronary artery bypass graft (CABG) surgery were assigned randomly to a RIPre group (n=20) or coronary heart disease (CHD) group (n=20). Twenty patients scheduled for mastectomy were enrolled as a control group. RIPre was achieved by occluding arterial blood flow 5 min with a mercury sphygmomanometer followed by a 5-min reperfusion period, and this was repeated 4 times. The RIPre procedure was repeated 3 times a day for 20 days. In all patients, arterial fragments discarded during surgery were collected to evaluate endothelial function by flow-mediated dilation (FMD), CD34+ monocyte count, and endothelial nitric oxide synthase (eNOS expression). Phosphorylation levels of STAT-3 and Akt were also assayed to explore the underlying mechanisms. Compared with the CHD group, long-term regular RIPre significantly improved FMD after 20 days (8.5±2.4 vs 4.9±4.2%, P<0.05) and significantly reduced troponin after CABG surgery (0.72±0.31 and 1.64±0.19, P<0.05). RIPre activated STAT-3 and increased CD34+ endothelial progenitor cell counts found in arteries. Long-term, regular RIPre improved endothelial function in patients with CHD, possibly due to STAT-3 activation, and this may have led to an increase in endothelial progenitor cells.
PMCID: PMC4470317  PMID: 25923462
Remote ischemic preconditioning; endothelial function; coronary heart disease; STAT-3
3.  Identification of biomarkers for lymph node metastasis in early-stage cervical cancer by tissue-based proteomics 
British Journal of Cancer  2014;110(7):1748-1758.
Pelvic lymph node metastasis (PLNM) is the key to determining the treatment and prognosis of early-stage cervical cancer (CC, I–IIst). The aim of this study was to identify biomarkers for PLNM of CC, I–IIst.
Two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to identify differentially expressed proteins in primary CC, I–IIst tissue with (n=8) and without (n=10) PLNM. The expression levels of three differential proteins (FABP5, HspB1, and MnSOD) were validated using western blotting and immunohistochemistry. An independent cohort of 105 CC, I–IIst patients was analysed to assess the correlation of FABP5, HspB1, and MnSOD with clinicopathologic factors and clinical outcomes.
Forty-one differential proteins were identified. Upregulation of FABP5, HspB1, and MnSOD in CC, I–IIst with PLNM was confirmed and was significantly correlated with PLNM. FABP5, HspB1, and MnSOD were significant predictors of PLNM in univariate analysis. FABP5, HspB1, and lymphovascular space invasion (LVSI) were independent predictors of PLNM in multivariate analysis. Survival curves indicated that CC, I–IIst patients with FABP5, HspB1, and MnSOD upregulation had poor prognosis.
FABP5, HspB1, and MnSOD may be potential biomarkers for PLNM of CC, I–IIst and may have important roles in the pathogenesis of PLNM.
PMCID: PMC3974096  PMID: 24569473
cervical cancer; proteomics; biomarkers; diagnosis; pelvic lymph node; metastasis
4.  Expression of insulin-like growth factor-1 receptor in keloid and hypertrophic scar 
Keloid and hypertrophic scar (HS) are two pathological forms of excessive dermal fibrosis, which are due to aberrant wound-healing responses. Accumulating evidence suggests that aberrant activity of growth factors and increased numbers of growth factor receptors play an important role in the formation of pathological scar.
We examined the expression level of insulin-like growth factor-1 receptor (IGF-IR) in keloid, HS and normal skin.
IGF-IR expression was analyzed by immunohistochemistry, real-time PCR and western blotting on tissues and fibroblasts from 30 patients, comprising 10 patients with keloid and 20 with HS (10 with immature and 10 with mature HS), and from 10 age-matched and sex-matched healthy controls.
Immunoreactivity to IGF-IR was found in dermal fibroblasts of keloid (90%), immature HS, (80%) and mature HS (30%), but not in normal skin. There was no statistically significant difference in immunoreactivity scores between keloid and immature HS, but there was a significant difference (P < 0.01) between mature and immature HS. Real-time PCR and western blot analysis confirmed that there was high expression of IGF-IR in keloid and immature HS fibroblasts, but not in mature HS or normal skin fibroblasts. IGF-IR was expressed in the overlying epidermis, and there was no significant difference between the groups.
IGF-IR may be involved in the pathogenesis of keloid and HS. Given that IGF-IR are predominantly expressed on dermal fibroblasts, targeting of IGF-IR in fibroblasts may be of benefit to prevent scarring.
PMCID: PMC4232319  PMID: 25154292
5.  The N-terminal region of p27 inhibits HIF-1α protein translation in ribosomal protein S6-dependent manner by regulating PHLPP-Ras-ERK-p90RSK axis 
Zhang, D | Liu, J | Mi, X | Liang, Y | Li, J | Huang, C
Cell Death & Disease  2014;5(11):e1535-.
P27 was identified as a tumor suppressor nearly two decades, being implicated in cell-cycle control, differentiation, senescence, apoptosis and motility. Our present study, for the first time to the best of our knowledge, revealed a potential role of p27 in inhibiting S6-mediated hypoxia-inducible factor-1α (HIF-1α) protein translation, which contributed to the protection from environmental carcinogen (sodium arsenite)-induced cell transformation. Our findings showed that depletion of p27 expression by knockout and knockdown approaches efficiently enhanced S6 phosphorylation in arsenite response via overactivating Ras/Raf/MEK/ERK pathway, which consequently resulted in the stimulation of p90RSK (90 kDa ribosomal S6 kinase), a direct kinase for S6 phosphorylation. Although PI3K/AKT pathway was also involved in S6 activation, blocking AKT and p70S6K activation did not attenuate arsenite-induced S6 activation in p27−/− cells, suggesting p27 specifically targeted Ras/ERK pathway rather than PI3K/AKT pathway for inhibition of S6 activation in response to arsenite exposure. Further functional studies found that p27 had a negative role in cell transformation induced by chronic low-dose arsentie exposure. Mechanistic investigations showed that HIF-1α translation was upregulated in p27-deficient cells in an S6 phosphorylation-dependent manner and functioned as a driving force in arsenite-induced cell transformation. Knockdown of HIF-1α efficiently reversed arsenite-induced cell transformation in p27-depleted cells. Taken together, our findings provided strong evidence showing that by targeting Ras/ERK pathway, p27 provided a negative control over HIF-1α protein synthesis in an S6-dependent manner, and abrogated arsenite-induced cell transformation via downregulation of HIF-1α translation.
PMCID: PMC4260754  PMID: 25412313
6.  A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia 
p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.
PMCID: PMC4244677  PMID: 25387678
Apoptosis; Chronic myeloid leukemia; p15INK4B; STI571 (Gleevec, imatinib)
7.  Latexin sensitizes leukemogenic cells to gamma-irradiation-induced cell-cycle arrest and cell death through Rps3 pathway 
Cell Death & Disease  2014;5(10):e1493-.
Leukemia is a leading cause of cancer death. Recently, the latexin (Lxn) gene was identified as a potential tumor suppressor in several types of solid tumors and lymphoma, and Lxn expression was found to be absent or downregulated in leukemic cells. Whether Lxn functions as a tumor suppressor in leukemia and what molecular and cellular mechanisms are involved are unknown. In this study, the myeloid leukemogenic FDC-P1 cell line was used as a model system and Lxn was ectopically expressed in these cells. Using the protein pull-down assay and mass spectrometry, ribosomal protein subunit 3 (Rps3) was identified as a novel Lxn binding protein. Ectopic expression of Lxn inhibited FDC-P1 growth in vitro. More surprisingly, Lxn enhanced gamma irradiation-induced DNA damages and induced cell-cycle arrest and massive necrosis, leading to depletion of FDC-P1 cells. Mechanistically, Lxn inhibited the nuclear translocation of Rps3 upon radiation, resulting in abnormal mitotic spindle formation and chromosome instability. Rps3 knockdown increased the radiation sensitivity of FDC-P1, confirming that the mechanism of action of Lxn is mediated by Rps3 pathway. Moreover, Lxn enhanced the cytotoxicity of chemotherapeutic agent, VP-16, on FDC-P1 cells. Our study suggests that Lxn itself not only suppresses leukemic cell growth but also potentiates the cytotoxic effect of radio- and chemotherapy on cancer cells. Lxn could be a novel molecular target that improves the efficacy of anti-cancer therapy.
PMCID: PMC4237263  PMID: 25341047
8.  Serum-dependent processing of late apoptotic cells for enhanced efferocytosis 
Cell Death & Disease  2014;5(5):e1264-.
Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.
PMCID: PMC4047901  PMID: 24874736
complement component C1q; phagocytosis; serum; efferocytosis
9.  Can early postoperative intraocular pressure predict success following mitomycin-C augmented trabeculectomy in primary angle-closure glaucoma 
Eye  2013;27(3):403-409.
To evaluate the association between early and late postoperative intraocular pressure (IOP) and determine if early postoperative IOP can predict the surgical outcome.
A total of 165 consecutive patients with primary angle-closure glaucoma (PACG) undergoing primary mitomycin-C-augmented trabeculectomy underwent a comprehensive eye examination before surgery and were followed-up on days 1, 7, 14, and 30, and months 3, 6, 12, and 18. IOPs on days 1, 7, 14, and 30 were stratified into groups A (<10 mm Hg), B (≥10 and <15 mm Hg), C (≥15 and <20 mm Hg), and D (≥20 mm Hg). Differences between groups were analyzed using analysis of variance (ANOVA) and Fisher's exact test. Multivariable regression was used to exam the predictive ability of early IOP for final outcome.
The mean age was 62.5±7.9 years and 41.21% (n=68) were males. Stratified by IOP on days 1, 7, 14, and 30, respectively, mean IOPs at month 18 were different among groups A, B, C, and D (ANOVA, P=0.047, P=0.033, P=0.008, and P<0.001, respectively). Once the IOPs were settled with interventions on day 7 a higher IOP level was associated with decreasing success rate under different outcome definitions, final IOP <15 mm Hg (Fisher's exact P=0.001) and <20 mm Hg (P=0.039) without medication. Multiple regression showed early IOP predicted final IOP independently from baseline variables. A cutoff value of 13.5 mm Hg on day 7 achieved an accuracy of 80.0 and 57.1% in predicting IOP<15 mm Hg without medication and failure after surgery, respectively.
The IOP at 18 months following primary antifibrotic-augmented trabeculectomy in PACG patients is associated with and predicted by the postoperative IOPs at 1 month. Control of early IOP to 13.5 or less may provide better outcomes.
PMCID: PMC3597889  PMID: 23306726
trabeculectomy; intraocular pressure; releasable sutures; outcome prediction; active bleb management; angle-closure glaucoma
10.  Fasting blood glucose is a novel prognostic indicator for extranodal natural killer/T-cell lymphoma, nasal type 
Cai, Q | Luo, X | Liang, Y | Rao, H | Fang, X | Jiang, W | Lin, T | Lin, T | Huang, H
British Journal of Cancer  2013;108(2):380-386.
Extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKTL) is an aggressive disease with poor prognosis, requiring risk stratification. However, the prognosis of ENKTL is not fully defined and needs supplementation. We hypothesised that fasting blood glucose (FBG) may be a new prognostic factor for ENKTL.
We retrospectively analysed 130 patients newly diagnosed with ENKTL.
Both univariate analysis and multivariate analysis revealed that FBG >100 mg dl−1 was associated with a poor outcome. Patients with FBG >100 mg dl−1 at diagnosis had more adverse clinical features, achieved lower complete remission rates (P=0.003) and had worse overall survival (P<0.001) and progression-free survival (P<0.001) compared with low-FBG patients. Measurement of FBG was helpful in differentiating between low-risk patients using the International Prognostic Index (IPI) and Prognosis Index for peripheral T-cell lymphoma (PIT) scoring and patients in a different category using the Korean Prognostic Index (KPI) scores with different survival outcomes (P<0.05).
Our data suggest that measuring FBG levels at diagnosis is a novel, independent predictor of prognosis in ENKTL and helps to distinguish low-risk patients with poor survival, and this holds true in patients considered low-risk by IPI, PIT and KPI.
PMCID: PMC3566812  PMID: 23299534
fasting blood glucose; extranodal NK/T-cell lymphoma; prognosis
11.  Soil degradation and prevention in greenhouse production 
SpringerPlus  2013;2(Suppl 1):S10.
Soil degradation has been a very serious problem for sustainable production, especially by a re-cropping of greenhouse-cultivated cucumber (Cucumis statirus L.). The aim of this research was to expound the actuality for soil degradation, at the same time, put forward some suggestion for preventing from soil degradation and maintain sustainable production in greenhouse basic on the two experiments conducted in a solar greenhouse during 2001-2008 suburb area of Yan'an, Shaanxi province in North China. The result shown that cucumber fruit productivity increased as the increasing of re-cropping years, but decreased after 5years continuously cropping. As increasing of re-cropping years, the population of fungus and bacteria increased, which was assumingly main factor of soil degradation. There was significant difference in cropping models on soil bio-characteristics and system productivity. The productivity were the highest in cropping model between cucumber and greengrocery, cucumber and cowpea (Vigna sinensis L), the second higher were in cropping model between cucumber and maize (Zea mays) for green manure, cucumber and kidney bean (Phaseolus vulgaris). That was the best way to reduce soil bacteria and epiphyte amount to follow lasting three or four months during summer season after cucumber harvest, the better method was planting cowpea or other leguminous crops. Basic on the experiment, the optimums approaches to preventive soil degradation were put forward.
PMCID: PMC3973405  PMID: 24701378
Soil degradation; greenhouse production; cropping model; soil bio-characteristics; preventing from soil degradation
12.  Ultrahigh relaxivity and safe probes of manganese oxide nanoparticles for in vivo imaging 
Scientific Reports  2013;3:3424.
Mn-based nanoparticles (NPs) have emerged as new class of probes for magnetic resonance imaging due to the impressive contrast ability. However, the reported Mn-based NPs possess low relaxivity and there are no immunotoxicity data regarding Mn-based NPs as contrast agents. Here, we demonstrate the ultrahigh relaxivity of water protons of 8.26 mM−1s−1 from the Mn3O4 NPs synthesized by a simple and green technique, which is twice higher than that of commercial gadolinium (Gd)-based contrast agents (4.11 mM−1s−1) and the highest value reported to date for Mn-based NPs. We for the first time demonstrate these Mn3O4 NPs biocompatibilities both in vitro and in vivo are satisfactory based on systematical studies of the intrinsic toxicity including cell viability of human nasopharyngeal carcinoma cells, normal nasopharyngeal epithelium, apoptosis in cells and in vivo immunotoxicity. These findings pave the way for the practical clinical diagnosis of Mn based NPs as safe probes for in vivo imaging.
PMCID: PMC4070373  PMID: 24305731
13.  Primary lymphoepithelioma-like carcinoma of ocular adnexa: clinicopathologic features and treatment 
Current Oncology  2013;20(2):e113-e122.
Background and Methods
Lymphoepithelioma-like carcinoma (lelc) is a rare malignancy in ocular adnexa.
Here, we report 4 patients with lelc and review 11 patients reported in the literature. Clinical profiles, association with Epstein–Barr virus (ebv), treatment, and outcomes are analyzed.
Lacrimal glands and the lacrimal drainage system, eyelid, and conjunctiva are potential primary sites for lelc. The tumours are characterized histologically by nests of undifferentiated malignant cells surrounded by lymphoid infiltrates. Infection with ebv was confirmed in lelc of ocular adnexa, and that association seemed to be restricted to Asian populations. Results from our centre uniformly showed expression of ebv-encoded small rnas in primary tumour, locally recurrent tumour, and metastatic lymph nodes. This disease had a tendency to relapse regionally. Postoperative radiotherapy seems to improve disease-free survival. Tumours appear to be sensitive to radiotherapy and chemotherapy based on cisplatin and 5-fluorouracil. At our centre, 3 patients were still living at 22, 33, and 76 months after surgery. One patient died of distant metastasis after a survival of 38 months.
Lymphoepithelioma-like carcinoma is a heterogenous entity among ocular adnexal malignancies. Multimodality treatment provides a better chance at survival. Further investigation is required to achieve a better understanding of the biologic behavior of this entity and of its optimal treatment.
PMCID: PMC3615862  PMID: 23559878
Lymphoepithelioma-like carcinoma; ocular adnexa; Epstein–Barr virus; surgery; radiotherapy
14.  GABAB-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction 
Translational Psychiatry  2012;2(7):e142-.
Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30–80 Hz) oscillatory abnormalities, phenotypes common to these disorders. However, circuit-level mechanisms underlying such deficits remain unclear. This study investigated the relationship between gamma synchrony, excitatory–inhibitory (E/I) signaling, and behavioral phenotypes in NMDA-NR1neo−/− mice, which have constitutively reduced expression of the obligate NR1 subunit to model disrupted developmental NMDAR function. Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABAB-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits. These data demonstrate a clinically relevant, highly translatable neural-activity-based biomarker for preclinical screening and therapeutic development across a broad range of disorders that share common endophenotypes and disrupted NMDA-receptor signaling.
PMCID: PMC3410621  PMID: 22806213
animal model; GABAergic signaling; gamma oscillation; neuropsychiatric disease; NMDA-receptor
16.  Molecular Characterization and Expression Pattern of Gene IGFBP-5 in the Cashmere Goat (Capra hircus) 
Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth, apoptosis and other IGF-stimulated signaling pathways. In order to explore the significance of IGFBP-5 in cells of the Inner Mongolian Cashmere goat (Capra hircus), IGFBP-5 gene complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the animal’s fetal fibroblasts and tissue-specific expression analysis was performed by semi-quantitative RT-PCR. The gene is 816 base pairs (bp) in length and includes the complete open reading frame, encoding 271 amino acids (GenBank accession number JF720883). The full cDNA nucleotide sequence has a 99% identity with sheep, 98% with cattle and 95% with human. The amino acids sequence shares identity with 99%, 99% and 99%, respectively. The bioinformatics analysis showed that IGFBP-5 has an insulin growth factor-binding protein homologues (IB) domain and a thyroglobulin type-1 (TY) domain, four protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, three prenyl group binding sites (CaaX box). The IGFBP-5 gene was expressed in all the tested tissues including testis, brain, liver, lung, mammary gland, spleen, and kidney, suggesting that IGFBP-5 plays an important role in goat cells.
PMCID: PMC4093108  PMID: 25049603
Cashmere Goat; IGFBP-5; Expression Pattern; Tissue-specific; Hair; Gene
Neuroscience  2006;141(3):1257-1264.
People with schizophrenia display sensory encoding deficits across a broad range of electrophysiological and behavioral measures, suggesting fundamental impairments in the ability to transduce the external environment into coherent neural representations. This inability to create basic components of complex stimuli interferes with a high fidelity representation of the world and likely contributes to cognitive deficits. The current study evaluates the effects of constitutive forebrain activation of the Gsα G-protein subunit on auditory threshold and gain using acoustic brainstem responses and cortically generated N40 event-related potentials to assess the role of cyclic AMP signaling in sensory encoding. Additionally, we examine the ability of pharmacological treatments that mimic (amphetamine) or ameliorate (haloperidol) positive symptoms of schizophrenia to test the hypothesis that the encoding deficits observed in Gsα transgenic mice can be normalized with treatment. We find that Gsα transgenic mice have decreased amplitude of cortically generated N40 but normal acoustic brainstem response amplitude, consistent with forebrain transgene expression and a schizophrenia endophenotype. Transgenic mice also display decreased stimulus intensity response (gain) in both acoustic brainstem response and N40, indicating corticofugal influence on regions that lack transgene expression. N40 deficits in transgenic animals were ameliorated with low dose haloperidol and reversed with higher dose, suggesting dopamine D2 receptor-linked Gi activity contributes to the impairment. Consistent with this hypothesis, we recreated the Gsα transgenic deficit in wild type animals using the indirect dopamine agonist amphetamine. This transgenic model of sensory encoding deficits provides a foundation for identifying biochemical contributions to sensory processing impairments associated with schizophrenia.
PMCID: PMC3311921  PMID: 16750890
Gsα; N40; schizophrenia; cAMP; haloperidol; event-related potentials
18.  In-vivo activity of dihydroartemisinin against Schistosoma japonicum 
PMCID: PMC4084662  PMID: 21396254
19.  Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice 
Neurobiology of disease  2010;40(3):503-517.
Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinson’s Disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human mutant wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.
PMCID: PMC2955774  PMID: 20659558
Parkinson’s Disease; Transgenic; Dopamine; Microdialysis; Neuropathology; Anxiety
20.  Toll-like receptors regulate B cell cytokine production in patients with diabetes 
Diabetologia  2010;53(7):1461-1471.
Understanding cellular and molecular events in diabetes mellitus will identify new approaches for therapy. Immune system cells are important modulators of chronic inflammation in diabetes mellitus, but the role of B cells is not adequately studied. The aim of this work was to define the function of B cells in diabetes mellitus patients through focus on B cell responses to pattern recognition receptors.
We measured expression and function of Toll-like receptors (TLRs) on peripheral blood B cells from diabetes mellitus patients by flow cytometry and multiplexed cytokine analysis. We similarly analysed B cells from non-diabetic donors and periodontal disease patients as comparative cohorts.
B cells from diabetes mellitus patients secrete multiple pro-inflammatory cytokines, and IL-8 production is significantly elevated in B cells from diabetic patients compared with those from non-diabetic individuals. These data, plus modest elevation of TLR surface expression, suggest B cell IL-8 hyperproduction is a cytokine-specific outcome of altered TLR function in B cells from diabetes mellitus patients. Altered TLR function is further evidenced by demonstration of an unexpected, albeit modest ‘anti-inflammatory’ function for TLR4. Importantly, B cells from diabetes mellitus patients fail to secrete IL-10, an anti-inflammatory cytokine implicated in inflammatory disease resolution, under a variety of TLR-stimulating conditions. Comparative analyses of B cells from patients with a second chronic inflammatory disease, periodontal disease, indicated that some alterations in B cell TLR function associate specifically with diabetes mellitus.
Altered TLR function in B cells from diabetes mellitus patients increases inflammation by two mechanisms: elevation of pro-inflammatory IL-8 and lack of anti-inflammatory/protective IL-10 production.
PMCID: PMC2895399  PMID: 20383694
B lymphocytes; Cytokines; Diabetes mellitus; Human; Toll-like receptors
21.  Differential expression of ryanodine receptor in the developing rat cochlea 
Ryanodine receptors (RyRs) are one of the intracellular calcium channels involved in regulation of intracellular free calcium concentration ([Ca2+]i). The immunolocalization of RyRs was investigated in the developing rat cochlea at different postnatal days (PND). The change of [Ca2+]i in isolated outer hair cells (OHCs) was determined. Morphological results showed low expression of RyRs in the Kolliker’s organ from the PND 5 group. RyR expression in inner hair cells (IHCs) increased as the rats aged, and was mature after PND 14. RyRs in OHCs were expressed near the synaptic area of afferent and efferent nerves. RyRs in supporting cells were expressed widely and strongly. The application of ACh, ryanodine + ACh, and thapsigargin + ACh could induce a significant increase in [Ca2+]i in OHCs in the presence of extracellular calcium. This increase of [Ca2+]i induced by ACh was caused by not only the calcium influx through surface calcium channels, but also the calciuminduced calcium release (CICR) from intracellular RyR-sensitive calcium stores. Morphological and Ca imaging results suggested that RyRs expression is related to cochlear maturity, and may play an important role in its function.
PMCID: PMC3167338  PMID: 22073362
ryanodine receptor; development of cochlea; Ca2+; calcium-induced calcium release.
22.  Defining myopia using refractive error and uncorrected logMAR visual acuity >0.3 from 1334 Singapore school children ages 7–9 years 
To determine the association of spherical equivalent (SE) with low uncorrected visual acuity (VA) along with a proposed definition for myopia using logMAR VA >0.3 as the criteria.
1334 Chinese schoolchildren (mean age 7.8; range 7–9 years) were enrolled in the study after those who had hyperopia ⩾+2.00 dioptres (D) and astigmatism > = −2.00D were excluded. Uncorrected logMAR VA was measured for both eyes. Cycloplegia autorefraction was achieved by the instillation of three drops of 1% cyclopentolate 5 minutes apart. The average of five successful consecutive refraction and keratometry readings were obtained with calibrated Canon RK5 autokeratorefractometers by well trained optometry students, at least 30 minutes after the instillation of the third drop of cyclopentolate. SE cut‐off points (−0.25D, −0.5D, −0.75D, −1.0D) were evaluated.
Using different SE cut‐off points, the myopia prevalence rates of this sample of schoolchildren varied from 45.8% (SE at least −0.25 D) to 30.7% (SE at least −1.0 D). The cut‐off point of ⩾−0.75 D had a sensitivity and specificity of 91.8% (95% CI, 89.2 to 94.4) and 93.7% (95% CI, 92.1 to 95.3), respectively, to predict low vision defined as uncorrected logMAR VA > 0.3 (either eye). The next best cut‐off point of −0.5D had a higher sensitivity (93.3%), but lower specificity (87.9%).
The cut‐off points of −0.75D and −0.5D in SE refraction are appropriate for the prediction of uncorrected logMAR VA worse than 0.3, which is the criterion for the US common state adult driver licensing standard.
PMCID: PMC1856974  PMID: 16488963
myopia; children; Singapore
23.  CYP1B1 expression is induced by docetaxel: effect on cell viability and drug resistance 
British Journal of Cancer  2008;98(3):564-570.
The cytochrome P450 CYP1B1 is consistently overexpressed in tumour cells as compared to their normal counterparts, but its precise role in drug resistance is yet to be defined. It has been reported that transfection of CYP1B1 results in increased resistance to docetaxel in V79 cells (McFadyen et al, 2001). In this study, we analysed changes in expression of CYP1B1 mRNA associated with pulse selection of MCF-7 cells with docetaxel. Docetaxel-selected MCF-7 cells (MCF-7 Txt), which showed increased resistance to this drug as compared to parental MCF-7 cells, showed a noteworthy increase in CYP1B1 mRNA expression, paralleled by increased ethoxyresorufin-O-deethylase (EROD) activity levels. This effect was not observed in cisplatin- or adriamycin-selected MCF-7 cells, or in docetaxel-selected colon, lung or pancreatic carcinoma cells. Short-term treatment with docetaxel induced CYP1B1 mRNA expression in MDA 453 and BT-20 breast carcinoma cells, but not in MCF-7 cells. Transfection of MCF-7 Txt cells with CYP1B1 siRNA did not significantly affect docetaxel-induced toxicity, but it decreased cell survival in the absence of drug. Preincubation of docetaxel with recombinant CYP1B1 did not affect drug toxicity in A549 cells. These results suggest that CYP1B1 does not directly inactivate docetaxel, but rather might promote cell survival in MCF-7 Txt cells, providing an explanation for its association with drug resistance.
PMCID: PMC2243158  PMID: 18212750
cytochromes P450; CYP1B1; drug resistance; breast
24.  Prostamides (prostaglandin-ethanolamides) and their pharmacology 
British Journal of Pharmacology  2007;153(3):410-419.
The prostamides are part of a large and continually expanding series of pharmacologically unique neutral lipids. They are COX-2 derived oxidation products of the endocannabinoid/endovanniloid anandamide. Prostamide pharmacology is unique and, as in the case of the endocannabinoids anandamide and 2-arachidonylglycerol, bears little resemblance to that of the corresponding free acids. By virtue of its close relationship to the anti-glaucoma drug bimatoprost, prostamide F2α has received the greatest research attention. Prostamide F2α and bimatoprost effects appear independent of prostanoid FP receptor activation, according to a litany of agonist studies. Studies involving freshly isolated and separate feline iridial smooth muscle cells revealed that bimatoprost and FP receptor agonists stimulated different cells, without exception. This suggests the existence of receptors that preferentially recognize prostamide F2α. The recent discovery of prostamide antagonists has provided further support for prostamide receptors as discrete entities. The prototypical prostamide antagonists, AGN 204396 and 7, blocked the effects of prostamide F2α and bimatoprost but not those of PGF2α and FP receptor agonists in the feline iris. Second generation more potent prostamide antagonists, such as AGN 211334, should allow the role of prostamides in health and disease to be elucidated. From the therapeutics standpoint, the prostamide F2α analogue bimatoprost is the most efficacious ocular hypotensive agent currently available for the treatment of glaucoma.
PMCID: PMC2241799  PMID: 17721551
prostaglandins; prostaglandin-ethanolamides; FP receptor; prostaglandin glyceryl ester; anandamide; cyclo-oxygenase-2
25.  In vitro and in vivo demonstration of risperidone implants in mice 
Schizophrenia research  2007;98(1-3):66-78.
Nonadherence with medication is a critical limitation in current long-term treatment of schizophrenia and a primary factor in poor quality-of-life outcomes. However, few treatments have addressed this shortcoming using an implantable drug delivery approach. The goal of this study was to provide in vitro and in vivo proof of concept for a long-term implantable risperidone delivery system in mice.
Implantable formulations of risperidone were created using the biodegradable polymer Poly Lactic co Glycolic Acid (PLGA) combined with various drug loads. Implant bioactivity was tested using in vitro release and stability studies, as well as in vivo pharmacokinetic and behavioral studies in mice.
The pattern of risperidone release is influenced by various parameters, including polymer composition and drug load. In vitro measures demonstrate that risperidone is stable in implants under physiological conditions. Behavioral measures demonstrate the bioactivity of risperidone implants delivering 3mg/kg/day in mice, while pharmacokinetic analyses indicate that reversibility is maintained throughout the delivery interval.
The current report suggests that implantable formulations are a viable approach to providing long-term delivery of antipsychotic medications based on in vivo animal studies and pharmacokinetics. Implantable medications demonstrated here can last two months or longer while maintaining coherence and removability past full release, suggesting a potential paradigm shift in the long-term treatment of schizophrenia.
PMCID: PMC2561216  PMID: 17765477
Treatment adherence; schizophrenia; risperidone pharmacokinetics; drug delivery system; drug implant; in vitro/in vivo correlation

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