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1.  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
2.  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.
doi:10.1111/acel.12134
PMCID: PMC3838489  PMID: 23834718
Notch1 signaling pathway; aged rat brain; neurogenesis; focal cerebral ischemia
3.  Neurogenesis in Adult Human Brain after Traumatic Brain Injury 
Journal of Neurotrauma  2013;30(22):1872-1880.
Abstract
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.
doi:10.1089/neu.2010.1579
PMCID: PMC3815038  PMID: 21275797
brain trauma; human; injury; neurogenesis; stem cells
4.  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.
doi:10.1111/jcmm.12115
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signaling
5.  Re-emergence of Rabies in the Guangxi Province of Southern China 
Background
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.
Conclusions
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.
doi:10.1371/journal.pntd.0003114
PMCID: PMC4183421  PMID: 25275567
6.  Pharmacokinetic Compatibility of Ginsenosides and Schisandra Lignans in Shengmai-san: From the Perspective of P-Glycoprotein 
PLoS ONE  2014;9(6):e98717.
Background
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.
Methodology
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.
doi:10.1371/journal.pone.0098717
PMCID: PMC4055595  PMID: 24922060
7.  Testing two models describing how methylome-wide studies in blood are informative for psychiatric conditions 
Epigenomics  2013;5(4):367-377.
Aim
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.
Conclusion
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.
doi:10.2217/epi.13.36
PMCID: PMC3904748  PMID: 23895651
antipsychotic; DNA methylation; methyl-CpG binding domain; methylome-wide association study; mirror-site model; next-generation sequencing; signature model
8.  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.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1007/s12325-014-0120-1
PMCID: PMC4033813  PMID: 24831915
Adherence; Cost; Hypoglycemia; Insulin detemir; Insulin glargine; Persistence; Switching; Type 2 diabetes
9.  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.
doi:10.1016/j.neurobiolaging.2012.10.029
PMCID: PMC3570710  PMID: 23182805
VEGF; Alzheimer’s disease; Human; brain; expression
10.  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)
11.  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.
doi:10.1111/jcmm.12201
PMCID: PMC4000112  PMID: 24597656
primary open angle glaucoma; exome sequencing; Chinese population
12.  MBD-seq as a cost-effective approach for methylome-wide association studies: demonstration in 1500 case–control samples 
Epigenomics  2012;4(6):605-621.
Aim
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.
Results
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.
Conclusion
This study showed the potential of MBD-seq as a cost-effective tool in large-scale disease studies.
doi:10.2217/epi.12.59
PMCID: PMC3923085  PMID: 23244307
MBD; methylome-wide association studies; next-generation sequencing; principal component analysis; pyrosequencing
13.  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.
doi:10.1371/journal.pone.0086625
PMCID: PMC3900578  PMID: 24466178
14.  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.
doi:10.1111/jcmm.12115
PMCID: PMC3877925  PMID: 24373503
Notch4; AVM; human; brain; signalling
15.  Real-world outcomes of initiating insulin glargine-based treatment versus premixed analog insulins among US patients with type 2 diabetes failing oral antidiabetic drugs 
Background
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).
Methods
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.
Results
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).
Conclusion
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.
doi:10.2147/CEOR.S49279
PMCID: PMC3794873  PMID: 24124384
type 2 diabetes mellitus; insulin glargine; rapid acting insulin; premixed insulin; clinical outcomes; treatment persistence
16.  Real world outcomes of adding rapid-acting insulin versus switching to analog premix insulin among US patients with type 2 diabetes treated with insulin glargine 
Introduction
Patients with type 2 diabetes mellitus (T2DM) often require intensification of basal insulin therapy. This retrospective, observational study compared real-world outcomes in US patients with T2DM treated with insulin glargine who added a rapid-acting insulin (RAI) (basal–bolus approach) with those who switched to premixed insulin (PMX).
Methods
The national US IMPACT® database was used to identify data from adult patients (≥18 years of age) with T2DM who added bolus RAI to insulin glargine (GLA + RAI) or who switched from GLA to PMX between 2001 and 2009. A stringent 1:1 propensity score-matching method was used to address the selection bias by matching GLA + RAI patients and PMX patients. Clinical and economic outcomes were determined for 1 year after the initial pharmacy claim for RAI or for PMX. Outcomes included treatment persistence and adherence, average insulin doses, glycated hemoglobin (A1C) levels, the prevalence and incidence of hypoglycemia, and health care costs/utilization. Analysis was carried out using an intent-to-treat approach.
Results
The study included data from 746 propensity-matched patients (n = 373 in each cohort). Treatment persistence and adherence were higher in the GLA + RAI cohort. There was no significant difference in A1C reduction from baseline and the number of patients achieving target A1C levels of <7% in each cohort. The incidence of hypoglycemic events was also similar in both groups. However, during follow-up, many patients (48.8%) who initially switched from insulin glargine to PMX crossed back over to use GLA and/or RAI as part of their regimen. Health care costs and utilization levels were not significantly different.
Conclusion
Clinical and economic outcomes were similar in T2DM patients who added RAI to GLA and in those who switched to PMX, but a basal–bolus strategy appears to be associated with better treatment persistence and adherence.
doi:10.2147/PPA.S49287
PMCID: PMC3786817  PMID: 24086105
type 2 diabetes mellitus; insulin glargine; rapid-acting insulin; premixed insulin; clinical outcomes; treatment persistence
17.  In Vitro Mesenchymal Trilineage Differentiation and Extracellular Matrix Production by Adipose and Bone Marrow Derived Adult Equine Multipotent Stromal Cells on a Collagen Scaffold 
Stem Cell Reviews  2013;9(6):858-872.
Directed differentiation of adult multipotent stromal cells (MSC) is critical for effective treatment strategies. This study was designed to evaluate the capability of equine MSC from bone marrow (BMSC) and adipose tissue (ASC) on a type I collagen (COLI) scaffold to undergo chondrogenic, osteogenic and adipogenic differentiation and form extracellular matrix (ECM) in vitro. Following determination of surface antigen expression, MSC were loaded into scaffolds in a perfusion bioreactor and loading efficiency was quantified. Cell-scaffold constructs were assessed after loading and 7, 14 and 21 days of culture in stromal or induction medium. Cell number was determined with DNA content, cell viability and spatial uniformity with confocal laser microscopy and cell phenotype and matrix production with light and scanning electron microscopy and mRNA levels. The MSC were positive for CD29 (>90 %), CD44 (>99 %), and CD105 (>60 %). Loading efficiencies were >70 %. The ASC and BMSC cell numbers on scaffolds were affected by culture in induction medium differently. Viable cells remained uniformly distributed in scaffolds for up to 21 days and could be directed to differentiate or to maintain an MSC phenotype. Micro- and ultrastructure showed lineage-specific cell and ECM changes. Lineage-specific mRNA levels differed between ASC and BMSC with induction and changed with time. Based on these results, equine ASC and BMSC differentiate into chondrogenic, osteogenic and adipogenic lineages and form ECM similarly on COLI scaffolds. The collected data supports the potential for equine MSC-COLI constructs to support diverse equine tissue formation for controlled biological studies.
doi:10.1007/s12015-013-9456-1
PMCID: PMC3834181  PMID: 23892935
Adipose; Bone marrow; Multipotent stromal cell; Equine; Extra-cellular matrix; Bioreactor; Osteogenesis; Adipogenesis; Chondrogenesis
18.  Synthesis and evaluation of [11C]MMPIP as a potential radioligand for imaging of metabotropic glutamate 7 receptor in the brain 
EJNMMI Research  2013;3:54.
Background
Metabotropic glutamate 7 (mGlu7) receptor is a crucial target protein for the development of pharmaceuticals against central nervous system disorders. In the present study, we synthesized [11C]MMPIP, a putative radioligand for mGlu7 (binding constant KB = 30 nM), and evaluated its potential for imaging of mGlu7 via in vitro and in vivo techniques.
Methods
[11C]MMPIP was synthesized by the reaction of phenol precursor 3 with [11C]CH3I. In vitro autoradiography using [11C]MMPIP was performed on rat brain sections. To determine in vitro specific binding of [11C]MMPIP with mGlu7, a blocking study was conducted by co-incubation with excess AMN082, a selective antagonist for mGlu7, or unlabeled MMPIP. Positron emission tomography (PET) studies and ex vivo metabolite analysis were carried out on rat brains.
Results
[11C]MMPIP was obtained with two specific activity (SA) levels of average 58 (conventional) and 3,800 (high SA) GBq/μmol, respectively. High radioactive signals derived from conventional [11C]MMPIP in the in vitro autoradiography were seen in the thalamus, medulla oblongata, and striatum, corresponding with comprehensive brain distributions of mGlu7. Co-incubation with ANM082 or unlabeled MMPIP reduced the radioactive signals in the brain sections, respectively. In the PET studies with [11C]MMPIP, no specific uptake relative to mGlu7 was found in the examined brain regions.
Conclusion
Despite in vitro specific binding of [11C]MMPIP with mGlu7, visualization of mGlu7 in the living brain using PET was not successful. Development of new ligand candidates with higher affinity for mGlu7 is necessary.
doi:10.1186/2191-219X-3-54
PMCID: PMC3751550  PMID: 23870677
MMPIP; mGlu7; PET; 11C; Autoradiography; Specific activity
19.  Relationships of hip joint volume ratios with degrees of joint laxity and degenerative disease from youth to maturity in a canine population predisposed to hip joint osteoarthritis 
Objective
To assess relationships of acetabular volume (AV), femoral head volume (FV), and portion of the femoral head within in the acetabulum (FVIA) with each other and with degrees of hip joint laxity and degenerative joint disease from youth to maturity in dogs predisposed to developing hip joint osteoarthritis (OA).
Animals
46 mixed-breed half- or full-sibling hound-type dogs.
Procedures
The distraction index (DI), AV, FV, FVIA, and degree of osteoarthritis (OA score) were quantified in 1 hip joint at 16, 32, and 104 weeks of age. Relationships among variables were evaluated within and between ages. Ratios corresponding to OA scores were compared within ages. Differences among 16-week ratios corresponding to 32-week OA scores and among 16- and 32-week ratios corresponding to 104-week OA scores were evaluated.
Results
Significant positive relationships existed between FV and AV across ages as well as between FVIA/FV and FVIA/AV and between DI and OA score across and within most ages. Such relationships also existed within these variables across most ages. Negative relationships of DI and OA scores with FVIA/FV and FVIA/AV within and among all ages were significant. Sixteen-week AVs, FVs, and FVIAs were greater and FV/AVs and OA scores were less than 32- and 104-week values. The 32-week FVIA/FV was less than 16- and 104-week values, and the 32-week FVIA/AV was less than the 104-week value. The FVIA/FV and FVIA/AV were lower and the DI was higher with higher OA scores within and among most ages.
Conclusions and Clinical Relevance
Structural volumes in lax canine hip joints changed predictably relative to each other during growth, despite degenerative changes. Measures developed in this study may augment current diagnosis and treatment strategies for hip dysplasia in dogs.
doi:10.2460/ajvr.72.3.376
PMCID: PMC3711502  PMID: 21355741
20.  Combined treatment of penile keloid: a troublesome complication after circumcision 
Asian Journal of Andrology  2013;15(4):575-576.
doi:10.1038/aja.2013.23
PMCID: PMC3739232  PMID: 23584379
21.  Interactions between Vascular Endothelial Growth Factor and Neuroglobin 
Neuroscience Letters  2012;519(1):47-50.
Vascular endothelial growth factor (VEGF) and neuroglobin (Ngb) participate in neuronal responses to hypoxia and ischemia, but the relationship between their effects, if any, is unknown. To address this issue, we measured Ngb levels in VEGF-treated mouse cerebrocortical cultures and VEGF levels in cerebrocortical cultures from Ngb-overexpressing transgenic mice. VEGF stimulated Ngb expression in a VEGFR2/Flk1 receptor-dependent manner, whereas Ngb overexpression suppressed expression of VEGF. These findings provide further insight into hypoxia-stimulated neuronal signaling pathways.
doi:10.1016/j.neulet.2012.05.018
PMCID: PMC3371093  PMID: 22583764
Vascular endothelial growth factor; neuroglobin; hypoxia
22.  Effect of a contralateral lesion on neurological recovery from stroke in rats 
Purpose
Clinical studies suggest a correlation between changes in activity of the contralesional cerebral cortex and spontaneous recovery from stroke, but whether this is a causal relationship is uncertain.
Methods
Young adult Sprague-Dawley male rats underwent unilateral or bilateral permanent distal middle cerebral artery occlusion (dMCAO). Infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining 24 hr after dMCAO, and functional outcome was assessed 1–28 days after dMCAO using the ladder rung walking and limb placing tests.
Results
Infarct volume was unchanged, but functional neurological deficits were reduced 1 day after bilateral compared to unilateral dMCAO.
Conclusions
Activity in the contralesional cerebral cortex may inhibit functional motor recovery acutely after experimental stroke.
doi:10.3233/RNN-2012-120254
PMCID: PMC3652379  PMID: 22868223
Stroke; ischemia; recovery; rat
23.  Treatment of MM-associated spinal fracture with percutaneous vertebroplasty (PVP) and chemotherapy 
European Spine Journal  2011;21(5):912-919.
Purpose
To investigate the effect of treatment of multiple myeloma (MM)-associated spinal fracture with percutaneous vertebroplasty (PVP) and chemotherapy.
Methods
Patients with MM-associated spinal fracture were randomly divided into combined (PVP and chemotherapy) treatment group (n = 38) and single chemotherapy group (n = 38). For the combined treatment group, bone cement was injected into vertebral body via DSA guided-percutaneous puncture. M2 scheme was used for both groups. And a 5-year follow-up was conducted for the study.
Results
At the 1-year follow-up visits, PVP combined with chemotherapy achieved complete remission (CR) in six patients (15.8%); near complete remission (nCR) in ten patients (26.30%); partial remission (PR) in nine patients (23.7%); minimal response (MR) in three patients (7.9%); no change (NC) in four patients (10.5%), and disease progression (DP) in five patients (13.2%). Only chemotherapy alone resulted in 3 CR (7.9%); 8 nCR (26.30%); 19 PR (77.5%); 4 MR (17.5%); 4 NC (17.5%), and 2 DP (5.0%). While the overall response rate (ORR) in the combined treatment group (65.8%) and the single chemotherapy group (50.0%) were significantly different, their visual analog pain scales (3.01 ± 0.62 and 5.97 ± 0.40, respectively) and Karnofsky performance scores (89.4 ± 6.3 and 80.3 ± 7.2, respectively) were significantly improved after treatment (P = 0.032 and P = 0.002, respectively). And the ORR between the two groups were significantly different (P = 0.001).
Conclusion
Percutaneous vertebroplasty is a minimally invasive surgery for MM-associated pathologic fracture. PVP had the characteristics of minimal trauma, easy operation and less complication. PVP can achieve long-term analgesic effect, and enhance the spinal stability.
doi:10.1007/s00586-011-2105-y
PMCID: PMC3337900  PMID: 22173608
Percutaneous puncture; Spine; Multiple myeloma; Fracture; Bone cement
24.  Vascular endothelial growth factor-B expression in postischemic rat brain 
Vascular Cell  2013;5:8.
Background
Vascular endothelial growth factor-B (VEGF-B) protects against experimental stroke, but the effect of stroke on VEGF-B expression is uncertain.
Methods
We examined VEGF-B expression by immunohistochemistry in the ischemic border zone 1–7 days after middle cerebral artery occlusion in rats.
Results
VEGF-B immunoreactivity in the border zone was increased after middle cerebral artery occlusion and was associated with neurons and macrophages/microglia, but not astrocytes or endothelial cells.
Conclusions
These findings provide additional evidence for a role of VEGF-B in the endogenous response to cerebral ischemia.
doi:10.1186/2045-824X-5-8
PMCID: PMC3671984  PMID: 23601533
Vascular endothelial growth factor-B (VEGF-B); Stroke; Ischemia
25.  High quality methylome-wide investigations through next-generation sequencing of DNA from a single archived dry blood spot 
Epigenetics  2013;8(5):542-547.
The potential importance of DNA methylation in the etiology of complex diseases has led to interest in the development of methylome-wide association studies (MWAS) aimed at interrogating all methylation sites in the human genome. When using blood as biomaterial for a MWAS the DNA is typically extracted directly from fresh or frozen whole blood that was collected via venous puncture. However, DNA extracted from dry blood spots may also be an alternative starting material. In the present study, we apply a methyl-CpG binding domain (MBD) protein enrichment-based technique in combination with next generation sequencing (MBD-seq) to assess the methylation status of the ~27 million CpGs in the human autosomal reference genome. We investigate eight methylomes using DNA from blood spots. This data are compared with 1,500 methylomes previously assayed with the same MBD-seq approach using DNA from whole blood. When investigating the sequence quality and the enrichment profile across biological features, we find that DNA extracted from blood spots gives comparable results with DNA extracted from whole blood. Only if the amount of starting material is ≤ 0.5µg DNA we observe a slight decrease in the assay performance. In conclusion, we show that high quality methylome-wide investigations using MBD-seq can be conducted in DNA extracted from archived dry blood spots without sacrificing quality and without bias in enrichment profile as long as the amount of starting material is sufficient. In general, the amount of DNA extracted from a single blood spot is sufficient for methylome-wide investigations with the MBD-seq approach.
doi:10.4161/epi.24508
PMCID: PMC3741224  PMID: 23644822
archived blood spots; methylation; next-generation sequencing; DNA extraction; MBD-seq

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