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1.  Genetic Variants Associated with Gestational Hypertriglyceridemia and Pancreatitis 
PLoS ONE  2015;10(6):e0129488.
Severe hypertriglyceridemia is a well-known cause of pancreatitis. Usually, there is a moderate increase in plasma triglyceride level during pregnancy. Additionally, certain pre-existing genetic traits may render a pregnant woman susceptible to development of severe hypertriglyceridemia and pancreatitis, especially in the third trimester. To elucidate the underlying mechanism of gestational hypertriglyceridemic pancreatitis, we undertook DNA mutation analysis of the lipoprotein lipase (LPL), apolipoprotein C2 (APOC2), apolipoprotein A5 (APOA5), lipase maturation factor 1 (LMF1), and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) genes in five unrelated pregnant Chinese women with severe hypertriglyceridemia and pancreatitis. DNA sequencing showed that three out of five patients had the same homozygous variation, p.G185C, in APOA5 gene. One patient had a compound heterozygous mutation, p.A98T and p.L279V, in LPL gene. Another patient had a compound heterozygous mutation, p.A98T & p.C14F in LPL and GPIHBP1 gene, respectively. No mutations were seen in APOC2 or LMF1 genes. All patients were diagnosed with partial LPL deficiency in non-pregnant state. As revealed in our study, genetic variants appear to play an important role in the development of severe gestational hypertriglyceridemia, and, p.G185C mutation in APOA5 gene appears to be the most common variant implicated in the Chinese population. Antenatal screening for mutations in susceptible women, combined with subsequent interventions may be invaluable in the prevention of potentially life threatening gestational hypertriglyceridemia-induced pancreatitis.
PMCID: PMC4469420  PMID: 26079787
2.  mTOR signaling inhibition modulates macrophages/microglia-mediated neuroinflammation and secondary injury via regulatory T cells after focal ischemia 
Signaling by the mammalian target of rapamycin (mTOR) plays an important role in the modulation of both innate and adaptive immune responses. However, the role and underlying mechanism of mTOR signaling in post-stroke neuroinflammation is largely unexplored. Here, we injected rapamycin, an mTOR inhibitor, by the intracerebroventricular route 6 hours after focal ischemic stroke in rats. We found that rapamycin significantly reduced lesion volume and improved behavioral deficits. Notably, infiltration of gamma delta T (γδ T) cells and granulocytes, which are detrimental to the ischemic brain, was profoundly reduced after rapamycin treatment, as was the production of pro-inflammatory cytokines and chemokines by macrophages and microglia. Rapamycin treatment prevented brain macrophage polarization towards the M1 type. In addition, we also found that rapamycin significantly enhanced anti-inflammation activity of regulatory T cells (Tregs), which decreased production of pro-inflammatory cytokines and chemokines by macrophages and microglia. Depletion of Tregs partially elevated macrophage/microglia-induced neuroinflammation after stroke. Our data suggest that rapamycin can attenuate secondary injury and motor deficits after focal ischemia by enhancing the anti-inflammation activity of Tregs to restrain post-stroke neuroinflammation.
PMCID: PMC4128178  PMID: 24829408
3.  Improved Visualization and Specific Binding for Metabotropic Glutamate Receptor Subtype 1 (mGluR1) Using [11C]ITMM with Ultra-High Specific Activity in Small-Animal PET 
PLoS ONE  2015;10(6):e0130006.
Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial target in the development of new medications to treat central nervous system (CNS) disorders. Recently, we developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[11C]methoxy-N-methyl-benzamide ([11C]ITMM) as a useful positron emission tomography (PET) probe for mGluR1 in clinical studies. Here, we aimed to improve visualization and threshold of specific binding for mGluR1 using [11C]ITMM with ultra-high specific activity (SA) of > 3,500 GBq/μmol in rat brains. A two-tissue compartment model indicated large differences between the two SAs in the constants k3 and k4, representing binding ability for mGluR1, while constants K1 and k2 showed no differences. The total distribution volume (VT) values of conventional and ultra-high SA were 9.1 and 11.2 in the thalamus, 7.7 and 9.7 in the striatum, and 6.4 and 8.5 mL/cm3 in the substantia nigra, respectively. The specific binding of [11C]ITMM with ultra-high SA was significantly higher than the conventional SA, especially in the basal ganglia. Parametric PET images scaled with VT of the ultra-high SA clearly identified regional differences in the rat brain. In conclusion, PET studies using [11C]ITMM with ultra-high SA could sufficiently improve visualization and specific binding for mGluR1, which could help further understanding for mGluR1 functions in CNS disorders.
PMCID: PMC4468202  PMID: 26076143
4.  Interference in the endplate nutritional pathway causes intervertebral disc degeneration in an immature porcine model 
International Orthopaedics  2014;38(5):1011-1017.
Previous studies have shown that blocking the endplate nutritional pathway with bone cement did not result in obvious intervertebral disc degeneration (IDD) in mature animal models. However, there are very few comparable studies in immature animal models. As vertebroplasty currently is beginning to be applied in young, even biologically immature patients, it is important to investigate the effect of cement blocking at the endplate in an immature animal model.
Two lumbar intervertebral discs in eight immature pigs were either blocked by cement in both endplate pathways or stabbed with a scalpel in the annulus fibrosus (AF) as a positive control, and with a third disc remaining intact as a normal control. Magnetic resonance imaging (MRI) and histology study were performed.
After three months, the cement-blocked discs exhibited severe IDD, with the percentage of disc-height index (DHI), nucleus pulposus (NP) area, and NP T2 value significantly lower than the normal control. These IDD changes were histologically confirmed. Post-contrast MRI showed diseased nutritional diffusion patterns in the cement-blocked discs. Moreover, the degenerative changes of the cement-blocked discs exceeded those of the injured AF positive controls.
The endplate nutritional pathway was interfered with and diseased after three months of bone cement intervention in an immature porcine model. Severe interference in the endplate nutritional pathway in an immature porcine model caused IDD. These findings also draw attention to the fact that interference in endplate nutritional pathways in immature or young patients may affect the vitality of adjacent discs.
PMCID: PMC3997759  PMID: 24652423
Endplate nutritional pathway; Intervertebral disc degeneration; Vertebroplasty; Bone cement; MRI; Immature porcine model
5.  Noninvasive quantification of metabotropic glutamate receptor type 1 with [11C]ITDM: a small-animal PET study 
Because of its role in multiple central nervous system (CNS) pathways, metabotropic glutamate receptor type 1 (mGluR1) is a crucial target in the development of pharmaceuticals for CNS disorders. N-[4-[6-(isopropylamino)-pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methyl-4-[11C]-methylbenzamide ([11C]ITDM) was recently developed as a positron emission tomography (PET) ligand for mGluR1. To devise a method for measurement of the binding potential (BPND) of [11C]ITDM to mGluR1, reference tissue methods aimed at replacing measurement of the arterial input function are desirable. In this study, we evaluated a noninvasive quantification method of mGluR1 with [11C]ITDM, demonstrating its accuracy using Huntington disease model R6/2 mice. The BPND measurements based on the Logan reference (Logan Ref) method have closely approximated that based on the arterial input method. We performed PET analysis with Logan Ref to assess its accuracy in quantifying the decline of mGluR1 expression in R6/2 mice. Significant decreases in BPND values in R6/2 mice were detected in cerebellum, thalamus, striatum, and cingulate cortex. We compared autoradiographs of R6/2 mouse brain sections with immunohistochemical images, and found a close correlation between changes in radioactive signal intensity and degree of mGluR1 expression. In conclusion, [11C]ITDM-PET is a promising tool for in vivo quantification of mGluR1 expression.
PMCID: PMC3982087  PMID: 24398932
BPND; [11C]ITDM; Huntington disease; mGluR1; positron emission tomography; R6/2
6.  Status and prospects of percutaneous vertebroplasty combined with 125I seed implantation for the treatment of spinal metastases 
Metastatic spinal tumours are the most common type of bone metastasis. Various methods have been used to treat metastatic spinal lesions, including radiotherapy, chemotherapy, isotope therapy, bisphosphonate therapy, analgesics, and surgery. Conservative treatments such as radiotherapy and chemotherapy are not appropriate and usually are ineffective in patients with vertebral fractures and/or spinal instability. Minimally invasive surgical treatments using non-vascular interventional technology, such as percutaneous vertebroplasty (PVP), have been successfully performed in the clinical setting. PVP is a non-invasive procedure that creates small wounds and is usually associated with only minor complications. In the present study, we will review the clinical status and prospects for the use PVP combined with 125I seed implantation (PVPI) to treat spinal metastases. The scientific evidence for this treatment, including safety, efficacy, and outcome measures, as well as comparisons with other therapies, was analysed in detail. PVPI effectively alleviates pain in metastatic spinal tumour patients, and the use of interstitial 125I seed implants can enhance the clinical outcomes. In conclusion, PVPI is a safe, reliable, effective, and minimally invasive treatment. The techniques of PVP and 125I seed implantation complement each other and strengthen the treatment’s effect, presenting a new alternative treatment for spinal metastases with potentially wide application.
PMCID: PMC4397861  PMID: 25886442
125I Seed implantation; Bone cement; Metastatic tumour; Percutaneous vertebroplasty; Spine
7.  A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects 
Human Molecular Genetics  2013;23(5):1175-1185.
The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25–92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10−8). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10−30). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.
PMCID: PMC3919012  PMID: 24135035
8.  Why Sn doping significantly enhances the dielectric properties of Ba(Ti1-xSnx)O3 
Scientific Reports  2015;5:8606.
Through appropriate doping, the properties of BaTiO3-based ferroelectrics can be significantly enhanced. To determine the physical process induced by the doping of Sn atoms in Ba(Ti0.8Sn0.2)O3, we performed high-resolution scanning transmission electron microscopy experiments and observed that the regions with low Sn content formed polar nano regions (PNRs) embedded in the matrix in Ba(Ti0.8Sn0.2)O3. The interactions among Sn, Ti, Ba and O atoms were determined using first principles calculations. Based on the characteristics of the electronic structure and crystal lattice strain fields, the effects of doping with Sn were investigated. The Sn doping not only changed the electronic structure of the crystal but also increased the dielectric properties of the PNRs. Moreover, the Sn doping was also responsible for the diffuse phase transition of the Ba(Ti1-xSnx)O3 material. The effects mentioned in this paper are universal in lead-free ferroelectrics, and similar elements such as Sb, Mg, and Zr may have the same functions in other systems. Thus, these results provide guidance for the design of the doping process and new systems of ferroelectric or relaxor materials.
PMCID: PMC4342585  PMID: 25721479
9.  Mechanism of immunosuppressants combined with cord blood for severe aplastic anemia 
This study aims to explore the mechanism of immunosuppressants combined with cord blood (IS + CBI) for severe aplastic anemia. Selecting 30 patients with SAA and all treated with IS + CBI (newly diagnosed group). 23 patients who were treated effectively (effective group) while 7 cases were treated invalidly (invalid group). Another 20 healthy individuals were selected as control group. To detect the expression levels of IL-17, IL-22 and other cytokines by ELISA method in each group. To detect the engraftment of cord blood stem cells by using short tandem repeat-polymerase chain reaction (STR-PCR) method. 1. IL-17, IL-22 and other cytokines expressions in newly diagnosed group were significantly higher than in the control group. 2. After 6 months, the level in effective group was significantly lower than pretherapy (P < 0.05).The level in invalid group had no obvious difference than pretherapy. 3. After 1 month and 3 months of treatment, a small amount of engraftment was found in effective group. After 6 months, implant rejection was showed. No effective engraftment was observed in invalid group. 1. IL-17, IL-22 cells in SAA patients increased which might positively correlated with the progression of SAA. 2. During the treatment of IS + CBI, there is a bridging mechanism between the early stage of engraftment and the advanced stage of immunosuppressant adjustment. The first 3 months after treatment, it relies on the engraftment of cord blood stem cells to promote hematopoietic recovery and 3 months later, it relies on immunosuppressants to maintain normal hematopoietic function.
PMCID: PMC4402841  PMID: 25932194
Aplastic anemia; cord blood; cytokine; interleukin-17; interleukin-22
10.  [18F]FEBMP: Positron Emission Tomography Imaging of TSPO in a Model of Neuroinflammation in Rats, and in vitro Autoradiograms of the Human Brain 
Theranostics  2015;5(9):961-969.
We evaluated the efficacy of 2-[5-(4-[18F]fluoroethoxy-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-methyl-N-phenylacetamide] ([18F]FEBMP) for positron emission tomography (PET) imaging of translocator protein (18 kDa, TSPO). Dissection was used to determine the distribution of [18F]FEBMP in mice, while small-animal PET and metabolite analysis were used for a rat model of focal cerebral ischemia. [18F]FEBMP showed high radioactivity uptake in mouse peripheral organs enriched with TSPO, and relatively high initial brain uptake (2.67 ± 0.12% ID/g). PET imaging revealed an increased accumulation of radioactivity in the infarcted striatum, with a maximum ratio of 3.20 ± 0.12, compared to non-injured striatum. Displacement with specific TSPO ligands lowered the accumulation levels in infarcts to those on the contralateral side. This suggests that the increased accumulation reflected TPSO-specific binding of [18F]FEBMP in vivo. Using a simplified reference tissue model, the binding potential on the infarcted area was 2.72 ± 0.27. Metabolite analysis in brain tissues showed that 83.2 ± 7.4% and 76.4 ± 2.1% of radioactivity was from intact [18F]FEBMP at 30 and 60 min, respectively, and that this ratio was higher than in plasma (8.6 ± 1.9% and 3.9 ± 1.1%, respectively). In vitro autoradiography on postmortem human brains showed that TSPO rs6971 polymorphism did not affect binding sites for [18F]FEBMP. These findings suggest that [18F]FEBMP is a promising new tool for visualization of neuroinflammation.
PMCID: PMC4493534  PMID: 26155312
[18F]FEBMP; translocator protein (18 kDa); neuroinflammation; binding potential; rs6971 polymorphism
11.  PET imaging of apoptosis in tumor-bearing mice and rabbits after paclitaxel treatment with 18F-Labeled recombinant human His10-annexin V 
Monitoring response to chemo- or radiotherapy is of great importance in clinical practice. Apoptosis imaging serves as a very useful tool for the early evaluation of tumor response. The goal of this study was PET imaging of apoptosis with 18F-labeled recombinant human annexin V linked with 10 histidine tag (18F-rh-His10-annexin V) in nude mice bearing an A549 tumor and rabbits bearing a VX2 lung cancer after paclitaxel therapy. 18F-rh-His10-annexin V was prepared by conjugation of rh-His10-annexin V with N-succinimidyl 4-[18F]fluorobenzoate. Biodistribution was determined in mice by the dissection method and small-animal PET. Single-dose paclitaxel (175 mg/m2) was used to induce apoptosis in A549 and VX2 tumor models. 18F-rh-His10-annexin V was injected into A549 mice and VX rabbits to acquire dynamic and static PET images 72 h after paclitaxel treatment. The uptake of 18F-rh-His10-annexin V in apoptotic cells 4 h after induction was 6.45±0.52 fold higher than that in non-induced cells. High focal uptake of 18F-rh-His10-annexin V was visualized in A549 (SUVmax: 0.35±0.13) and VX2 (0.41±0.23) tumor models after paclitaxel treatment, whereas lower uptake was found in the corresponding tumors before treatment (A549 SUVmax: 0.04±0.02; VX2: 0.009±0.002). The apoptotic index was 75.61±11.56% in the treated VX2 cancer, much higher than that in the untreated VX2 (8.03±2.81%). This study demonstrated the feasibility of 18F-rh-His10-annexin V for the detection of apoptosis after chemotherapy in A549 and VX2 tumor models.
PMCID: PMC4299778  PMID: 25625024
Apoptosis; molecular imaging; recombinant human His10-annexin V; tumor response
12.  Notch1 Signaling Modulates Neuronal Progenitor Activity in the Subventricular Zone in Response to Aging and Focal Ischemia 
Aging cell  2013;12(6):10.1111/acel.12134.
Neurogenesis diminishes with aging and ischemia-induced neurogenesis also occurs, but reduced in aged brain. Currently, the cellular and molecular pathways mediating these effects remain largely unknown. Our previous study has shown that Notch1 signaling regulates neurogenesis in subventricular zone (SVZ) of young-adult brain after focal ischemia, but whether a similar effect occurs in aged normal and ischemic animals is unknown. Here, we used normal and ischemic aged rat brains to investigate whether Notch1 signaling was involved in the reduction of neurogenesis in response to aging and modulates neurogenesis in aged brains after focal ischemia. By Western blot, we found that Notch1 and Jagged1 expression in the SVZ of aged brain was significantly reduced compared with young-adult brain. Consistently, the activated form of Notch1(Notch intracellular domain;NICD) expression was also declined. Immunohistochemistry confirmed that expression and activation of Notch1 signaling in the SVZ of aged brain were reduced. Double or triple immunostaining showed that that Notch1 was mainly expressed in DCX-positive cells, whereas Jagged1 was predominantly expressed in astroglial cells in the SVZ of normal aged rat brain. In addition, disruption or activation of Notch1 signaling altered the number of proliferating cells labeled by bromodeoxyuridine (BrdU) and doublecortin (DCX) in the SVZ of aged brain. Moreover, ischemia-induced cell proliferation in the SVZ of aged brain was enhanced by activating the Notch1 pathway, and was suppressed by inhibiting the Notch1 signaling. Reduced infarct volume and improved motor deficits were also observed in Notch1 activator-treated aged ischemic rats. Our data suggest that Notch1 signaling modulates the SVZ neurogenesis in aged brain in normal and ischemic conditions.
PMCID: PMC3838489  PMID: 23834718
Notch1 signaling pathway; aged rat brain; neurogenesis; focal cerebral ischemia
13.  Neurogenesis in Adult Human Brain after Traumatic Brain Injury 
Journal of Neurotrauma  2013;30(22):1872-1880.
While much work has been conducted regarding the neurogenesis response to traumatic brain injury (TBI) in rodents, it remains largely unknown whether neurogenesis in adult human brain also responds to TBI in a similar manner. Here, we performed immunocytochemistry on 11 brain specimens from patients with traumatic brain injury, who underwent surgical intervention. We found that expression of neural stem/progenitor cell (NSC) protein markers, including DCX, TUC4, PSA-NCAM, SOX2 and NeuroD, was increased in the perilesional cortex of human brain after TBI compared to that of normal brain. Confocal images showed that these NSC proteins were expressed in one single cell. We also found that proliferative markers were expressed in NSC protein-positive cells after TBI, and the number of proliferative NSCs was significantly increased after TBI. Our data suggest that TBI may also induce neurogenesis in human brain.
PMCID: PMC3815038  PMID: 21275797
brain trauma; human; injury; neurogenesis; stem cells
14.  Notch4 is Activated in Endothelial and Smooth Muscle Cells in Human Brain Arteriovenous Malformations 
Journal of cellular and molecular medicine  2013;17(11):10.1111/jcmm.12115.
Upregulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically-resection brain AVMs and found that Notch4 was upregulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch 4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signaling to the development of brain AVMs in human.
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signaling
15.  Re-emergence of Rabies in the Guangxi Province of Southern China 
Human rabies cases in the Guangxi province of China decreased from 839 in 1982 to 24 in 1995, but subsequently underwent a sharp increase, and has since maintained a high level.
Methodology/Principal Findings
3,040 brain samples from normal dogs and cats were collected from 14 districts of Guangxi and assessed by RT-PCR. The brain samples showed an average rabies virus (RV) positivity rate of 3.26%, but reached 4.71% for the period Apr 2002 to Dec 2003. A total of 30 isolates were obtained from normal dogs and 28 isolates from rabid animals by the mouse inoculation test (MIT). Six representative group I and II RV isolates showed an LD50 of 10−5.35/ml to 10−6.19/ml. The reactivity of monoclonal antibodies (MAbs) to group I and II RV isolates from the Guangxi major epidemic showed that eight anti-G MAbs showed strong reactivity with isolates of group I and II with titers of ≥10,000; however, the MAbs 9-6, 13-3 and 12-14 showed lower reactivity. Phylogenetic analysis based on the G gene demonstrated that the Guangxi RV isolates have similar topologies with strong bootstrap values and are closely bonded. Alignment of deduced amino acids revealed that the mature G protein has four substitutions A96S, L132F, N436S, and A447I specific to group I, and 13 substitutions T90M, Y168C, S204G, T249I, P253S, S289T, V332I, Q382H, V427I, L474P, R463K Q486H, and T487N specific to group II, coinciding with the phylogenetic analysis of the isolates.
Re-emergence of human rabies has mainly occurred in rural areas of Guangxi since 1996. The human rabies incidence rate increased is related with RV positive rate of normal dogs. The Guangxi isolates tested showed a similar pathogenicity and antigenicity. The results of phylogenetic analysis coincide with that of alignment of deduced amino acids.
Author Summary
Rabies is a worldwide zoonosis disease and is of considerable public health threat and hazard. The Guangxi province of southern China is a severe rabies epidemic region. Human rabies cases decreased from 839 in 1982 to 24 in 1995 in Guangxi as a result of a dog vaccination campaign. However, the number subsequently underwent a sharp increase, and has since maintained a high level. This study reports the systematic surveillance of rabies in Guangxi over the 30-year period from 1982 to 2012. The data revealed that a re-emergence of human rabies has occurred mainly in rural areas of Guangxi since 1996. Human rabies incidence rate increased follows increased instances of RV positive normal dogs. To further understand this re-emergence of rabies, the biological properties of the rabies virus (RV), including the RV-positive rate of normal dogs, pathogenicity, antigenicity and evolution, have been evaluated. The Guangxi isolates all showed similar pathogenicity and antigenicity. These isolates also exhibited similar topologies with strong bootstrap values in the two groups and were closely bonded. Thus these findings will be helpful to understanding the epidemiological situation for rabies in Guangxi.
PMCID: PMC4183421  PMID: 25275567
16.  Pharmacokinetic Compatibility of Ginsenosides and Schisandra Lignans in Shengmai-san: From the Perspective of P-Glycoprotein 
PLoS ONE  2014;9(6):e98717.
Phytochemical-mediated alterations in P-glycoprotein (P-gp) activity may result in herb-drug interactions by altering drug pharmacokinetics. Shengmai-san, a traditional Chinese herbal medicine composed by Panax Ginseng, Ophiopogon Japonicus, and Schisandra Chinensis, is routinely being used for treating various coronary heart diseases. In our previous studies, Schisandra Lignans Extract (SLE) was proved as a strong P-gp inhibitor, and herein, the compatibility of Shengmai-san was studied by investigating the influence of SLE on the pharmacokinetics of the ginsenosides from the perspective of P-gp.
Pharmacokinetic experiments were firstly performed based on in vitro uptake, efflux and transport experiments in Caco-2, LLC-PK1 wild-type and MDR1-overexpressing L-MDR1 cells. During the whole experiment, digoxin, a classical P-gp substrate, was used as a positive control drug to verify the cells used are the valid models. Meanwhile, the effects of SLE on the pharmacokinetics of ginsenosides were further investigated in rats after single-dose and multi-dose of SLE.
Results and Conclusions
The efflux ratios of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 were found more than 3.5 in L-MDR1 cells and can be decreased significantly by verapamil (a classical P-gp inhibitor). Contrarily, the efflux ratios of other ginsenosides (Rh1, F1, Re, and Rg1) were lower than 2.0 and not affected by verapamil. Then, the effects of SLE on the uptake and transport of ginsenosides were investigated, and SLE was found can significantly enhance the uptake and inhibit the efflux ratio of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 in Caco-2 and L-MDR1 cells. Besides, In vivo experiments showed that single-dose and multi-dose of SLE at 500 mg/kg could increase the area under the plasma concentration time curve of Rb2, Rc and Rd significantly without affecting terminal elimination half-time. In conclusion, SLE could enhance the exposure of ginsenosides Rb2, Rc, Rg2, Rg3, Rd and Rb1 significantly.
PMCID: PMC4055595  PMID: 24922060
17.  Testing two models describing how methylome-wide studies in blood are informative for psychiatric conditions 
Epigenomics  2013;5(4):367-377.
As the primary relevant tissue (brain) for psychiatric disorders is commonly not available, we aimed to investigate whether blood can be used as a proxy in methylation studies on the basis of two models. In the ‘signature’ model methylation–disease associations occur because a disease-causing factor affected methylation in the blood. In the ‘mirror-site’ model the methylation status in the blood is correlated with the corresponding disease-causing site in the brain.
Materials, methods & results
Methyl-binding domain enrichment and next-generation sequencing of the blood, cortex and hippocampus from four haloperidol-treated and ten untreated C57BL/6 mice revealed high levels of correlation in methylation across tissues. Despite the treatment inducing a large number of methylation changes, this correlation remains high.
Our results show that, consistent with the signature model, factors that affect brain processes (i.e., haloperidol) leave biomarker signatures in the blood and, consistent with the mirror-site model, the methylation status of many sites in the blood mirror those in the brain.
PMCID: PMC3904748  PMID: 23895651
antipsychotic; DNA methylation; methyl-CpG binding domain; methylome-wide association study; mirror-site model; next-generation sequencing; signature model
18.  Much Ado About Nothing? A Real-World Study of Patients with Type 2 Diabetes Switching Basal Insulin Analogs 
Advances in Therapy  2014;31(5):539-560.
Type-2 diabetes mellitus (T2DM) is a progressive disease, and many patients eventually require insulin therapy. This study examined real-world outcomes of switching basal insulin analogs among patients with T2DM.
Using two large United States administrative claims databases (IMPACT® and Humana®), this longitudinal retrospective study examined two cohorts of adult patients with T2DM. Previously on insulin glargine, Cohort 1 either continued insulin glargine (GLA-C) or switched to insulin detemir (DET-S), while Cohort 2 was previously on insulin detemir, and either continued insulin detemir (DET-C) or switched to insulin glargine (GLA-S). One-year follow-up treatment persistence and adherence, glycated hemoglobin (HbA1c), hypoglycemia events, healthcare utilization and costs were assessed. Selection bias was minimized by propensity score matching between treatment groups within each cohort.
A total of 5,921 patients (mean age 60 years, female 50.0%, HbA1c 8.6%) were included in the analysis (Cohort 1: IMPACT®: n = 536 DET-S matched to n = 2,668 GLA-C; Humana®: n = 256 DET-S matched to n = 1,262 GLA-C; Cohort 2: n = 419 GLA-S matched to n = 780 DET-C), with similar baseline characteristics between treatment groups in each cohort. During 1-year follow-up, in Cohort 1, DET-S patients, when compared with GLA-C patients, had lower treatment persistence/adherence with 33–40% restarting insulin glargine, higher rapid-acting insulin use, worse HbA1c outcomes, significantly higher diabetes drug costs, and similar hypoglycemia rates, health care utilization and total costs. However, in Cohort 2 overall opposite outcomes were observed and only 19.8% GLA-S patients restarted insulin detemir.
This study showed contrasting clinical and economic outcomes when patients with T2DM switched basal insulin analogs, with worse outcomes observed for patients switching from insulin glargine to insulin detemir and improved outcomes when switching from insulin detemir to insulin glargine. Further investigation into the therapeutic interchangeability of insulin glargine and insulin detemir in the real-world setting is needed.
Electronic supplementary material
The online version of this article (doi:10.1007/s12325-014-0120-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4033813  PMID: 24831915
Adherence; Cost; Hypoglycemia; Insulin detemir; Insulin glargine; Persistence; Switching; Type 2 diabetes
19.  Expression Level of Vascular Endothelial Growth Factor in Hippocampus is Associated with Cognitive Impairment in Patients with Alzheimer’s Disease 
Neurobiology of aging  2012;34(5):1412-1415.
Although the enhanced expression of VEGF in the brains of patients with Alzheimer’s disease (AD) has been reported, the functional significance of VEGF level in the progression of AD is still unclear. We examined the VEGF expression in the hippocampus of patients with AD at different stages of progression by Western blot, and found that VEGF (VEGF189) was barely detectable in normal hippocampus, but significantly increased at the early stage of patients with AD. VEGF189 was decreased with advancing stages of AD. Immunostaining shows that VEGF was significantly increased in the cells in the CA1, CA3 and dentate gyrus regions of hippocampus and the layer III and V of entorhinal cortex of patient with AD, compared to normal brain. Confocal images show that VEGF was predominantly expressed in neurons and astrocyte in the hippocampus and entorhinal cortex of patients with AD. Our data suggest that VEGF level is associated with progressive loss of cognitive function in patients with AD.
PMCID: PMC3570710  PMID: 23182805
VEGF; Alzheimer’s disease; Human; brain; expression
20.  PET brain kinetics studies of 11C-ITMM and 11C-ITDM,radioprobes for metabotropic glutamate receptor type 1, in a nonhuman primate 
The metabotropic glutamate receptor type 1 (mGluR1) is a novel target protein for the development of new drugs against central nervous system disorders. Recently, we have developed 11C-labeled PET probes 11C-ITMM and 11C-ITDM, which demonstrate similar profiles, for imaging of mGluR1. In the present study, we compared 11C-ITMM and 11C-ITDM PET imaging and quantitative analysis in the monkey brain. Respective PET images showed similar distribution of uptake in the cerebellum, thalamus, and cingulate cortex. Slightly higher uptake was detected with 11C-ITDM than with 11C-ITMM. For the kinetic analysis using the two-tissue compartment model (2-TCM), the distribution volume (VT) in the cerebellum, an mGluR1-rich region in the brain, was 2.5 mL∙cm-3 for 11C-ITMM and 3.6 mL∙cm-3 for 11C-ITDM. By contrast, the VT in the pons, a region with negligible mGluR1 expression, was similarly low for both radiopharmaceuticals. Based on these results, we performed noninvasive PET quantitative analysis with general reference tissue models using the time-activity curve of the pons as a reference region. We confirmed the relationship and differences between the reference tissue models and 2-TCM using correlational scatter plots and Bland-Altman plots analyses. Although the scattergrams of both radiopharmaceuticals showed over- or underestimations of reference tissue model-based the binding potentials against 2-TCM, there were no significant differences between the two kinetic analysis models. In conclusion, we first demonstrated the potentials of 11C-ITMM and 11C-ITDM for noninvasive PET quantitative analysis using reference tissue models. In addition, our findings suggest that 11C-ITDM may be superior to 11C-ITMM as a PET probe for imaging of mGluR1, because regional VT values in PET with 11C-ITDM were higher than those of 11C-ITMM. Clinical studies of 11C-ITDM in humans will be necessary in the future.
PMCID: PMC3999406  PMID: 24795840
Central nervous system (CNS); positron emission tomography (PET); metabotropic glutamate receptor type 1 (mGluR1)
21.  Family-based analysis identified CD2 as a susceptibility gene for primary open angle glaucoma in Chinese Han population 
Primary open angle glaucoma (POAG) is characterized by optic disc cupping and irreversible loss of retinal ganglion cells. Few genes have been detected that influence POAG susceptibility and little is known about its genetic architecture. In this study, we employed exome sequencing on three members from a high frequency POAG family to identify the risk factors of POAG in Chinese population. Text-mining method was applied to identify genes associated with glaucoma in literature, and protein–protein interaction networks were constructed. Furthermore, reverse transcription PCR and Western blot were performed to confirm the differential gene expression. Six genes, baculoviral inhibitors of apoptosis protein repeat containing 6 (BIRC6), CD2, luteinizing hormone/choriogonadotropin receptor (LHCGR), polycystic kidney and hepatic disease gene 1 (PKHD1), phenylalanine hydroxylase (PAH) and fucosyltransferase 7 (FUT7), which might be associated with POAG, were identified. Both the mRNA expression levels and protein expression levels of HSP27 were increased in astrocytes from POAG patients compared with those from normal control, suggesting that mutation in CD2 might pose a risk for POAG in Chinese population. In conclusion, novel rare variants detected by exome sequencing may hold the key to unravelling the remaining contribution of genetics to complex diseases such as POAG.
PMCID: PMC4000112  PMID: 24597656
primary open angle glaucoma; exome sequencing; Chinese population
22.  MBD-seq as a cost-effective approach for methylome-wide association studies: demonstration in 1500 case–control samples 
Epigenomics  2012;4(6):605-621.
We studied the use of methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) as a cost-effective screening tool for methylome-wide association studies (MWAS).
Materials & methods
Because MBD-seq has not yet been applied on a large scale, we first developed and tested a pipeline for data processing using 1500 schizophrenia cases and controls plus 75 technical replicates with an average of 68 million reads per sample. This involved the use of technical replicates to optimize quality control for multi- and duplicate-reads, an in silico experiment to identify CpGs in loci with alignment problems, CpG coverage calculations based on multiparametric estimates of the fragment size distribution, a two-stage adaptive algorithm to combine data from correlated adjacent CpG sites, principal component analyses to control for confounders and new software tailored to handle the large data set.
We replicated MWAS findings in independent samples using a different technology that provided single base resolution. In an MWAS of age-related methylation changes, one of our top findings was a previously reported robust association involving GRIA2. Our results also suggested that owing to the many confounding effects, a considerable challenge in MWAS is to identify those effects that are informative about disease processes.
This study showed the potential of MBD-seq as a cost-effective tool in large-scale disease studies.
PMCID: PMC3923085  PMID: 23244307
MBD; methylome-wide association studies; next-generation sequencing; principal component analysis; pyrosequencing
23.  Visualization of Acute Liver Damage Induced by Cycloheximide in Rats Using PET with [18F]FEDAC, a Radiotracer for Translocator Protein (18 kDa) 
PLoS ONE  2014;9(1):e86625.
Liver damage induced by drug toxicity is an important concern for both medical doctors and patients. The aim of this study was to noninvasively visualize acute liver damage using positron emission tomography (PET) with N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[18F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide ([18F]FEDAC), a radiotracer specific for translocator protein (18 kDa, TSPO) as a biomarker for inflammation, and to determine cellular sources enriching TSPO expression in the liver. A mild acute liver damage model was prepared by a single intraperitoneal injection of cycloheximide (CHX) into rats. Treatment with CHX induced apoptosis and necrotic changes in hepatocytes with slight neutrophil infiltration. The uptake of radioactivity in the rat livers was measured with PET after injection of [18F]FEDAC. The uptake of [18F]FEDAC increased in livers damaged from treatment with CHX compared to the controls. Presence of TSPO was examined in the liver tissue using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical assays. mRNA expression of TSPO was elevated in the damaged livers compared to the controls, and the level was correlated with the [18F]FEDAC uptake and severity of damage. TSPO expression in the damaged liver sections was mainly found in macrophages (Kupffer cells) and neutrophils, but not in hepatocytes. The elevation of TSPO mRNA expression was derived from the increase of the number of macrophages with TSPO and neutrophils with TSPO in damaged livers. From this study we considered that PET imaging with [18F]FEDAC represented the mild liver damage through the enhanced TSPO signal in inflammatory cells. We conclude that this method may be a useful tool for diagnosis in early stage of acute liver damage.
PMCID: PMC3900578  PMID: 24466178
24.  Notch4 is activated in endothelial and smooth muscle cells in human brain arteriovenous malformations 
Up-regulation of Notch4 was observed in the endothelial cells in the arteriovenous malformations (AVMs) in mice. However, whether Notch4 is also involved in brain AVMs in humans remains unclear. Here, we performed immunohistochemistry on normal brain vascular tissue and surgically resected brain AVMs and found that Notch4 was up-regulated in the subset of abnormal vessels of the brain AVM nidus, compared with control brain vascular tissue. Two-photon confocal images show that Notch4 was expressed not only in the endothelial but also in the smooth muscle cells of the vascular wall in brain AVMs. Western blotting shows that Notch4 was activated in brain AVMs, but not in middle cerebral artery of normal human brain, which was confirmed by immunostaining. Our findings suggest a possible contribution of Notch4 signalling to the development of brain AVMs in human.
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signalling
25.  Real-world outcomes of initiating insulin glargine-based treatment versus premixed analog insulins among US patients with type 2 diabetes failing oral antidiabetic drugs 
In patients with type 2 diabetes mellitus, basal-bolus strategies can improve treatment by offering dosing flexibility, and improved satisfaction, adherence, and clinical outcomes. The purpose of this study was to compare real-world outcomes between US patients initiating analog insulin therapy with insulin glargine and those initiating with a premixed analog insulin (PMX).
This was a retrospective study of data from patients (≥18 years) with type 2 diabetes mellitus in the IMPACT® database who initiated insulin treatment with insulin glargine (GLA) or a PMX. Clinical and economic outcomes were measured over one year, including persistence and adherence, consumption of insulin, glycemic outcomes, incident hypoglycemia, and health care resource utilization and cost.
Data from 2,502 patients were included in the analyses (n = 834 for PMX, n = 1,668 for GLA). Compared with PMX, persistence was higher and consumption of insulin was lower for GLA (both P < 0.0001). Adherence, glycemic outcomes, and hypoglycemia-related events were similar between groups, as were health care utilization and total health care costs. Diabetes-related drug and supply costs were lower for GLA than for PMX (P < 0.0001 and P = 0.046, respectively).
In US patients with type 2 diabetes mellitus, initiating insulin with once-daily GLA, rather than a PMX, is associated with increased treatment persistence and similar clinical and hypoglycemic outcomes, but lower diabetes pharmacy and supply costs. GLA may be a more flexible option than PMX. However, these results also show suboptimal glycemic control in the real-world setting despite change in treatment regimens and call for optimization in management of patients with type 2 diabetes mellitus.
PMCID: PMC3794873  PMID: 24124384
type 2 diabetes mellitus; insulin glargine; rapid acting insulin; premixed insulin; clinical outcomes; treatment persistence

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