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1.  Puerarin and betahistine treatment of vertebrobasilar ischemia vertigo: A meta-analysis of randomized controlled trials 
The present meta-analysis aimed to evaluate the effectiveness and safety of puerarin co-treatment with betahistine in treating vertebrobasilar ischemia (VBI) vertigo. A total of 6 medical databases were searched, identifying randomized controlled trials (RCTs) of VBI vertigo performed until August 2014 that investigated a combined treatment of puerarin with betahistine or with other conventional drugs. The quality of the literature was evaluated using the Cochrane Collaboration's tool for assessing risk of bias, and Rev Man 5.0 software was used for statistical analysis and evaluation. The present study included 7 RCTs, involving a total of 664 subjects, and revealed a statistically significant increase in efficacy between the control and the experimental group (odds ratio [OR], 4.99; 95% confidence interval [CI], 3.05 to 8.15). The average blood flow velocity within the vertebrobasilar arteries increased following treatment with puerarin and betahistine compared with that of the control groups (OR, 7.59; 95% CI, 6.19 to 9.00); however, no difference was detected between these groups in the average flow velocity within the left vertebral artery (OR, 6.17; 95% CI, 5.22 to 7.13). The frequency of adverse reactions in the experimental group was lower (OR, 0.75; 95% CI, 0.32 to 1.77) compared with the control group. Combined puerarin and betahistine regimens were more effective in treating VBI vertigo compared with other, conventional drugs; effectively alleviating the associated symptoms, including dizziness and increased average blood flow velocity within the vertebrobasilar arteries, without causing an increased number of serious side effects. However, the efficacy and safety of puerarin and betahistine use in treating VBI vertigo requires additional investigation.
doi:10.3892/etm.2016.3004
PMCID: PMC4774609  PMID: 26998036
puerarin; betahistine; vertebrobasilar ischemia vertigo; randomized controlled trials; meta-analysis
2.  Novel cycloartane triterpenoid from Cimicifuga foetida (Sheng ma) induces mitochondrial apoptosis via inhibiting Raf/MEK/ERK pathway and Akt phosphorylation in human breast carcinoma MCF-7 cells 
Chinese Medicine  2016;11:1.
Background
Cycloartane triterpenoids exhibited anticancer effects. This study aims to identify any potential novel anticancer cycloartane triterpenoids from Cimicifuga foetida L. rhizome (Sheng ma) and the mode of actions.
Methods
Cycloartane triterpenoids were isolated from the C. foetida rhizome by a series of column chromatography and identified by IR, MS and NMR. Their anticancer effects on several human cancer cell lines, MCF-7, HepG2, HepG2/ADM, HeLa, and PC3, and normal human mammary epithelial cells MCF10A were investigated by colony formation and MTT assays. Morphological analysis of apoptosis induction was performed by acridine orange/ethidium bromide dual-staining and Hoechst 33258 nuclear staining. The cell-cycle profile and annexin V staining were evaluated by flow cytometry. Apoptosis were investigated by measuring changes in mitochondrial membrane potential and analyzing expression of cell cycle- and apoptosis-related proteins in MCF-7 cells by Western blotting.
Results
A novel cycloartane triterpenoid, 25-O-acetyl-7,8-didehydrocimigenol-3-O-β-d-(2-acetyl)xylopyranoside (ADHC-AXpn), together with the known 7,8-didehydrocimigenol-3-O-β-d-xylopyranoside (DHC-Xpn) were isolated. MCF-7 growth was significantly inhibited by ADHC-AXpn in a dose- and time-dependent manner (IC50: 27.81 µM at 48 h; P = 0.004 vs. control at 25 μM for 48 h treatment), and ADHC-AXpn was selectively cytotoxic for cancerous cells (MCF-7, HepG2/ADM, HepG2 and HELA cells) based on its higher IC50 values for normal cells MCF10A (IC50: 78.63 µM at 48 h) than for tumor cells. In MCF-7 cells, ADHC-AXpn induced G2/M cell cycle arrest by mediating cyclin-B1, and CDK1 and its phosphorylation; and induced apoptosis through the mitochondrial-mediated apoptotic pathway, with inhibition of Akt activation. As ADHC-AXpn suppressed phosphorylation of ERK1/2, Raf and Akt proteins in MCF-7 cells, its apoptotic effect might be associated with Raf/MEK/ERK signaling and Akt activation.
Conclusions
ADHC-AXpn significantly suppressed the growth of MCF-7 cells, induced mitochondrial apoptosis and cell-cycle arrest, and inhibited Raf/MEK/ERK signaling pathway and Akt phosphorylation.
doi:10.1186/s13020-015-0073-6
PMCID: PMC4709995  PMID: 26759603
4.  Arenobufagin intercalates with DNA leading to G2 cell cycle arrest via ATM/ATR pathway 
Oncotarget  2015;6(33):34258-34275.
Arenobufagin, a representative bufadienolide, is the major active component in the traditional Chinese medicine Chan'su. It possesses significant antineoplastic activity in vitro. Although bufadienolide has been found to disrupt the cell cycle, the underlying mechanisms of this disruption are not defined. Here, we reported that arenobufagin blocked the transition from G2 to M phase of cell cycle through inhibiting the activation of CDK1-Cyclin B1 complex; The tumor suppressor p53 contributed to sustaining arrest at the G2 phase of the cell cycle in hepatocellular carcinoma (HCC) cells. Moreover, arenobufagin caused double-strand DNA breaks (DSBs) and triggered the DNA damage response (DDR), partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. Importantly, we used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The hypochromicity in the UV-visible absorption spectrum, the significant changes in the circular dichroism (CD) spectrum of DNA, and the distinct quenching in the fluorescence intensity of the ethidium bromide (EB)-DNA system before and after arenobufagin treatment indicated that arenobufagin bound to DNA in vitro by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Collectively, these data provide novel insights into arenobufagin-induced cell cycle disruption that are valuable for the further discussion and investigation of the use of arenobufagin in clinical anticancer chemotherapy.
PMCID: PMC4741450  PMID: 26485758
arenobufagin; DNA intercalator; DNA damage response; G2 cell cycle arrest
5.  Indirect estimation of a discrete-state discrete-time model using secondary data analysis of regression data 
Statistics in medicine  2009;28(16):2095-2115.
SUMMARY
Multi-state models of chronic disease are becoming increasingly important in medical research to describe the progression of complicated diseases. However, studies seldom observe health outcomes over long time periods. Therefore, current clinical research focuses on the secondary data analysis of the published literature to estimate a single transition probability within the entire model. Unfortunately, there are many difficulties when using secondary data, especially since the states and transitions of published studies may not be consistent with the proposed multi-state model. Early approaches to reconciling published studies with the theoretical framework of a multi-state model have been limited to data available as cumulative counts of progression. This paper presents an approach that allows the use of published regression data in a multi-state model when the published study may have ignored intermediary states in the multi-state model. Colloquially, we call this approach the Lemonade Method since when study data give you lemons, make lemonade. The approach uses maximum likelihood estimation. An example is provided for the progression of heart disease in people with diabetes.
doi:10.1002/sim.3599
PMCID: PMC4621762  PMID: 19455575
diabetes; chronic disease; designed absorption; multi-state model; meta-analysis
6.  16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia 
Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.
PMCID: PMC4694369  PMID: 26770469
Ventilator-associated pneumonia (VAP); lower respiratory tract; oropharynx; 16S rRNA gene sequencing; 16S rDNA
7.  Phylogenetic reconstruction and polymorphism analysis of BK virus VP2 gene isolated from renal transplant recipients in China 
BK polyomavirus (BKV) is important pathogen for kidney transplant recipients, as it is frequently re-activated, leading to nephropathy. The aim of this study was to investigate the phylogenetic reconstruction and polymorphism of the VP2 gene in BKV isolated from Chinese kidney transplant recipients. Phylogenetic analysis was carried out in the VP2 region from 135 BKV-positive samples and 28 reference strains retrieved from GenBank. The unweighted pair-group method with arithmetic mean (UPGMA) grouped all strains into subtypes, but failed to subdivide strains into subgroups. Among the plasma and urine samples, all plasma (23/23) and 82 urine samples (82/95) were identified to contain subtype I; the other 10 urine samples contained subtype IV. A 86-bp fragment was identified as a highly conserved sequence. Following alignment with 36 published BKV sequences from China, 92 sites of polymorphism were identified, including 11 single nucleotide polymorphisms (SNPs) prevalent in Chinese individuals and 30 SNPs that were specific to the two predominant subtypes I and IV. The limitations of the VP2 gene segment in subgrouping were confirmed by phylogenetic analysis. The conserved sequence and polymorphism identified in this study may be helpful in the detection and genotyping of BKV.
doi:10.3892/etm.2015.2723
PMCID: PMC4665150  PMID: 26640547
BK polyomavirus; VP2 gene; genotypes; polymorphism; Chinese
8.  Crude triterpenoid saponins from Ilex latifolia (Da Ye Dong Qing) ameliorate lipid accumulation by inhibiting SREBP expression via activation of AMPK in a non-alcoholic fatty liver disease model 
Chinese Medicine  2015;10:23.
Background
Ilex latifolia Thunb. (Da Ye Dong Qing) is used for weight loss and for its antidiabetic effects. This study aims to investigate the beneficial effects and potential mechanisms of action of crude triterpenoid saponins (CTS) from I. latifolia in a mouse model of high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD).
Methods
Male C57BL/6 mice (n = 50), were arbitrarily divided into five groups (n = 10 in each group): a control group, HFD group, simvastatin group (10 mg/kg/day), and two CTS treatment groups (100 and 200 mg/kg/day). All mice except those in the control group were fed an HFD for 4 weeks. Animals in the treatment groups were orally administered simvastatin or CTS for 8 weeks. Oral glucose tolerance tests and insulin tolerance tests were performed. At the end of treatment, plasma lipid levels, and oxidative parameters in the liver were measured using commercial test kits. Western blotting was used to evaluate whether CTS induced AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase activation, and the expression of transcription factors and their target genes was evaluated in a quantitative PCR assay.
Results
Compared with the HFD group, the CTS (200 mg/kg/day) treatment group showed significantly decreased plasma lipid parameters (P < 0.001, P = 0.018, and P = 0.005 for triglycerides, total cholesterol and low-density lipoprotein cholesterol, respectively), and improved insulin resistance (P = 0.006). CTS (100 and 200 mg/kg/day) supplementation also reduced hepatic lipids and protected the liver from oxidative stress by attenuating malondialdehyde content (P < 0.001 and P < 0.001, respectively) and restoring aspartate aminotransferase levels (P < 0.001 and P < 0.001, respectively). Moreover, CTS (200 mg/kg/day) reduced lipid accumulation by enhancing AMPK phosphorylation and inhibiting expression of sterol regulatory element-binding proteins (SREBPs) and their target genes SREBP-1c, SREBP-2, fatty acid synthase, and stearoyl-CoA desaturase (P = 0.013, P = 0.007, P = 0.011, and P = 0.014, respectively).
Conclusion
CTS from I. latifolia improved insulin resistance and liver injury in HFD-fed mice, and attenuated NAFLD via the activation of AMPK and inhibition of the gene expression of SREBPs and some of their target molecules.
doi:10.1186/s13020-015-0054-9
PMCID: PMC4544818  PMID: 26300958
9.  Apolipoprotein E gene E2/E2 genotype is a genetic risk factor for vertebral fractures in humans: a large-scale study 
International Orthopaedics  2014;38(8):1665-1669.
Purpose
Although many studies have been performed to evaluate whether or not apolipoprotein E gene (APOE) polymorphisms are differentially associated with bone mineral density (BMD) and fractures, the results have been conflicting. This large-scale study was performed to investigate whether a relationship exists between APOE polymorphisms and risk of fracture.
Methods
A hospital-based case–control study was conducted in 3,000 patients with fractures and 3,000 age- and gender-matched healthy controls. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay was applied to assess the APOE gene polymorphisms.
Results
Patients with fractures had a significantly higher frequency of APOE E2/E2 genotype [odds ratio (OR) = 2.02, 95 % confidence interval (CI) = 1.30, 3.14; P = 0.002] than healthy controls. When stratifying by fracture type, it was found that patients with vertebral fractures had a significantly higher frequency of APOE E2/E2 genotype (OR = 2.86, 95 % CI = 1.73, 4.73; P < 0.001). No significant differences were found in nonvertebral (hip or wrist or other) fractures.
Conclusions
Our study suggests that APOE E2/E2 genotype is a potential genetic risk factor for vertebral fractures in humans.
doi:10.1007/s00264-014-2380-4
PMCID: PMC4115114  PMID: 24880936
Apolipoprotein E; Fracture; Gene polymorphism; Case–control study
10.  Crude triterpenoid saponins from Anemone flaccida (Di Wu) exert anti-arthritic effects on type II collagen-induced arthritis in rats 
Chinese Medicine  2015;10:20.
Background
Anemone flaccida Fr . Schmidt (Ranunculaceae) (Di Wu in Chinese) is used to treat punch injury and rheumatoid arthritis (RA). However, the active compounds and underlying mechanism of action mediating the anti-arthritic effects of A. flaccida remain unclear. This study aims to evaluate the underlying action mechanism of A. flaccida crude triterpenoid saponins (AFS) on RA using a type II collagen (CII)-induced arthritis (CIA) rat model, and to assess the anti-inflammatory effects of the main active compounds of AFS, namely flaccidoside II, anhuienoside E, glycoside St-I4a, hemsgiganoside B, hederasaponin B, and 3-O-α-l-rhamnopyranosyl (1 → 2)-β-d-glucopyranosyl oleanolic acid 28-O-β-d-glucopyranosyl (1 → 6)-β-d-glucopyranosyl ester.
Methods
Male Wistar rats (n = 50) were randomly separated into five groups (n = 10) and immunized by CII injection. AFS (200 or 400 mg/kg) and dexamethasone were orally administered for 30 days after establishing the model. The arthritis severity was assessed by paw volume using a plethysmometer. After 30 days of treatment, the right hind paws of the rats were obtained. Paw histology was analyzed by hematoxylin and eosin staining, and radiologic imaging was performed by micro-computed tomography. MTT assays were used to evaluate the cytotoxicity of AFS and its main compounds in RAW264.7 cells. Enzyme-linked immunosorbent assay kits were used to measure interleukin (IL)-6 and tumor necrosis factor (TNF)-α in serum and supernatants from AFS- and main AFS compound-treated RAW264.7 cells stimulated by lipopolysaccharide (LPS).
Results
Anemone flaccida crude triterpenoid saponins inhibited redness and swelling of the right hind paw in the CIA model. Radiological and histological examinations indicated that inflammatory responses were reduced by AFS treatment. Moreover, comparing with untreated rats, serum TNF-α (P = 0.0035 and P < 0.001) and IL-6 (P = 0.0058 and P = 0.0087) were lower in AFS-treated CIA rats at the dose of 200 and 400 mg/kg/day. AFS and its main compounds, including hederasaponin B, flaccidoside II, and hemsgiganoside B, significantly inhibited TNF-α (P = 0.0022, P = 0.013, P = 0.0015, and P = 0.016) and IL-6 (P = 0.0175, P < 0.001, P < 0.001, and P < 0.001) production in LPS-treated RAW264.7 cells, respectively.
Conclusions
Anemone flaccida crude triterpenoid saponins and its main bioactive components, including hederasaponin B, flaccidoside II, and hemsgiganoside B, decreased pro-inflammatory cytokine levels in a CIA rat model and LPS-induced RAW264.7 cells.
doi:10.1186/s13020-015-0052-y
PMCID: PMC4515010  PMID: 26213566
11.  Warfarin dosage adjustment strategy in Chinese population 
Background: Blood anticoagulation after heart valve replacement is a recognized difficulty all over the world. In this study, we identified the effect of amiodarone on the function of warfarin and confirmed the countermeasure by concluding the genotype distribution of vitamin K epoxide reductase complex 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) of the patient to predict the security dose of warfarin. Methods: Studying on the VKORC1 (-1639G>A) and CYP2C9 genotype of 271 cases on heart valve replacement in the First Affiliated Hospital of Soochow University from Jan. 2012 to Jan. 2014. Warfarin’s multivariable regression equation was taken to calculate their warfarin dosage. In the study, 80 of them were selected and divided into 4 groups according to their different warfarin dosage and their usage of amiodaron. The differences of INR values at the 5th, 8th, 11th, 14th days of operation were analyzed. Results: Among the 80 cases, VKORC1 (-1639G>A) AA types accounted for 90%, and AG types accounted for another 10%, while GG types were not found. In addition that, all of the patients (100%) had CYP2C9*1/*1 type, and CYP2C9*1/*3 had not appeared. There was significant difference in INR values between the groups who used amiodarone or not. The pharmacogenetic equation was accurate in the predicting of the warfarin dosage, so that satisfied anticoagulation efficacy had been achieved in 2 weeks after surgery. Conclusion: It is necessary for the patients to do the warfarin pharmacogenetic test to get the suitable dose before heart valve replacement. Amiodarone can enhance the anticoagulant efficacy of warfarin, so the dosages of warfarin should be reduced properly because of the medicine combination, and INR values must be monitored more frequently to make the anticoagulant process secure and efficient.
PMCID: PMC4538110  PMID: 26309674
Heart valve replacement surgery; anticoagulation; pharmacogenomic testing; warfarin; amiodarone
12.  Outflow facility efficacy of five drugs in enucleated porcine eyes by a method of constant-pressure perfusion 
This study aimed to characterize a technique that assesses the outflow facility (C) efficacy of five kinds of IOP-lowering drugs commonly used clinically in enucleated porcine Eyes. Eyes were perfused at 15 mmHg with GPBS first to establish the baseline outflow facility (C0). Then the anterior chamber contents were exchanged for GPBS with corresponding concentration eye drops (4.9×103 nM Brimonidine, 41.1 nM Latanoprost, 3.4×103 nM Levobunolol, 3.0×103 nM Brinzolamide, 8.3×103 nM Pilocarpine) in five groups (n = 6 each), while 6 eyes received GPBS alone as control. The mean stable facility obtained after drug administration (C1) was continuously recorded. The changes between C0 and C1 (ΔC = C1-C0) were analyzed. Finally, for drugs among the five experiment groups with statistical significance, the concentration was reduced 3 times, otherwise the drugs’ concentration was increased to 10 times to confirm its effectiveness further using the same methods (n = 6 each). We found that the average baseline outflow facility was 0.24±0.01 μl·min-1·mmHg-1. C increased significantly in Brimonidine and Latanoprost groups, even the concentration of Brimonidine and Latanoprost was decreased 3 times (P < 0.05). However, there was no significantly increase in Levobunolol, Brinzolamide, Pilocarpine and control group (P > 0.05), but when drugs’ concentration was increased to 10 times, the C value of Pilocarpine decreased significantly (P = 0.04). No significant washout effects in porcine eyes were observed. To conclude, outflow facility efficacy of five drugs in enucleated porcine eyes may provide a reference for clinical medicine. A constant-pressure perfusion technique should be useful to evaluate effect of pharmacologic agents or surgical manipulations on aqueous humor dynamics.
PMCID: PMC4509202  PMID: 26221257
Aqueous humor outflow facility; ocular perfusion; porcine eyes; ocular hypotensive drugs
13.  Fanconi syndrome due to prolonged use of low-dose adefovir 
Fanconi syndrome results from a generalized abnormality of the proximal tubules of the kidney and owing to phosphate depletion can cause hypophosphatemic osteomalacia. Adefovir dipivoxyl (ADV) effectively suppresses hepatitis B virus replication but exhibits nephrotoxicity when administered at a low dosage. We report two cases of Fanconi syndrome induced by ADV at 10 mg/day to call for regular screening for evidence of proximal tubular dysfunction and detailed bone metabolic investigations for prompt detection of ADV nephrotoxicity is critically important to ensure timely drug withdrawal before the development of irreversible tubulointerstitial injury.
PMCID: PMC4468461  PMID: 26110001
Adefovir; Fanconi syndrome; hypophosphatemia; nephrotoxicity
14.  Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus 
Background
Very virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius.
Results
The viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels.
Conclusion
This study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.
doi:10.1186/s12917-015-0377-x
PMCID: PMC4395976  PMID: 25884204
vvIBDV; Viral load; GeXP; Real-time PCR; Pro-inflammatory cytokines; Chemokines
15.  Segmentations of MRI Images of the Female Pelvic Floor: A Study of Inter- and Intra-reader Reliability 
Purpose
To describe the inter- and intra-operator reliability of segmentations of female pelvic floor structures.
Materials and Methods
Three segmentation specialists were asked to segment out the female pelvic structures in 20 MR datasets on three separate occasions. The STAPLE algorithm was used to compute inter- and intra-segmenter agreement of each organ in each dataset. STAPLE computed the sensitivity, specificity, and positive predictive values (PPV) for inter- and intra-segmenter repeatability. These parameters were analyzed using intra-class correlation analysis. Correlation of organ volume to PPV and sensitivity was also computed.
Results
Mean PPV of the segmented organs ranged from 0.82 to 0.99, and sensitivity ranged from 33 to 96%. Intra-class correlation ranged from 0.07 to 0.98 across segmenters. Pearson correlation of volume to sensitivity were significant across organs, ranging from 0.54 to 0.91. Organs with significant correlation of PPV to volume were bladder (−0.69), levator ani (−0.68), and coccyx (−0.63).
Conclusion
Undirected manual segmentation of the pelvic floor organs are adequate for locating the organs, but poor at defining structural boundaries.
doi:10.1002/jmri.22478
PMCID: PMC4364418  PMID: 21563253
segmentation; MRI; pelvic floor muscles; Intra-class correlation; positive predictive value; repeatability
16.  The application of click chemistry in the synthesis of agents with anticancer activity 
The copper(I)-catalyzed 1,3-dipolar cycloaddition between alkynes and azides (click chemistry) to form 1,2,3-triazoles is the most popular reaction due to its reliability, specificity, and biocompatibility. This reaction has the potential to shorten procedures, and render more efficient lead identification and optimization procedures in medicinal chemistry, which is a powerful modular synthetic approach toward the assembly of new molecular entities and has been applied in anticancer drugs discovery increasingly. The present review focuses mainly on the applications of this reaction in the field of synthesis of agents with anticancer activity, which are divided into four groups: topoisomerase II inhibitors, histone deacetylase inhibitors, protein tyrosine kinase inhibitors, and antimicrotubule agents.
doi:10.2147/DDDT.S56038
PMCID: PMC4362898  PMID: 25792812
topoisomerase II inhibitors; histone deacetylase inhibitors; protein tyrosine kinase inhibitors; antimicrotubule agents
17.  Universal Stem-Loop Primer Method for Screening and Quantification of MicroRNA 
PLoS ONE  2014;9(12):e115293.
RT-qPCR is the accepted technique for the quantification of microRNA (miR) expression: however, stem-loop RT-PCR, the most frequently used method for quantification of miRs, is time- and reagent-consuming as well as inconvenient for scanning. We established a new method called ‘universal stem-loop primer’ (USLP) with 8 random nucleotides instead of a specific sequence at the 3′ end of the traditional stem-loop primer (TSLP), for screening miR profile and to semi-quantify expression of miRs. Peripheral blood samples were cultured with phytohaemagglutinin (PHA), and then 87 candidate miRs were scanned in cultured T cells. By USLP, our study revealed that the expression of miR-150-5p (miR-150) decreased nearly 10-fold, and miR-155-5p (miR-155) increased more than 7-fold after treated with PHA. The results of the dissociation curve and gel electrophoresis showed that the PCR production of the USLP and TSLP were specificity. The USLP method has high precision because of its low ICV (ICV<2.5%). The sensitivity of the USLP is up to 103 copies/µl miR. As compared with the TSLP, USLP saved 75% the cost of primers and 60% of the test time. The USLP method is a simple, rapid, precise, sensitive, and cost-effective approach that is suitable for screening miR profiles.
doi:10.1371/journal.pone.0115293
PMCID: PMC4280144  PMID: 25548906
18.  OTUB1 promotes metastasis and serves as a marker of poor prognosis in colorectal cancer 
Molecular Cancer  2014;13:258.
Background
OTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) is a deubiquitinating enzyme (DUB) that belongs to the OTU (ovarian tumor) superfamily. The aim of this study was to clarify the role of OTUB1 in colorectal cancer (CRC) and to identify the mechanism underlying its function.
Methods
Two hundred and sixty CRC samples were subjected to association analysis of OTUB1 expression and clinicopathological variables using immunohistochemical (IHC) staining. Overexpression of OTUB1 was achieved in SW480 and DLD-1 cells, and downregulation of OTUB1 was employed in SW620 cells. Then, migration and invasion assays were performed, and markers of the epithelial-mesenchymal transition (EMT) were analyzed. In addition, hepatic metastasis models in mice were used to validate the function of OTUB1 in vivo.
Results
OTUB1 was overexpressed in CRC tissues, and the expression level of OTUB1 was associated with metastasis. A high expression level of OTUB1 was also associated with poor survival, and OTUB1 served as an independent prognostic factor in multivariate analysis. OTUB1 also promoted the metastasis of CRC cell lines in vitro and in vivo by regulating EMT.
Conclusions
OTUB1 promotes CRC metastasis by facilitating EMT and acts as a potential distant metastasis marker and prognostic factor in CRC. Targeting OTUB1 may be helpful for the treatment of CRC.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-258) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-258
PMCID: PMC4351937  PMID: 25431208
OTUB1; Colorectal cancer; Metastasis; EMT; Prognostic factor
19.  Simultaneous determination by UPLC-MS/MS of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets: application to a pharmacokinetic study*  
A rapid, reliable, and sensitive method was developed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with an electrospray ionization (ESI) source for determination of seven bioactive compounds in rat plasma after oral administration of Ginkgo biloba tablets (GBTs). The method simultaneously detects bilobalide (BB), ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), quercetin (QCT), kaempferol (KMF), and isorhamnetin (ISR) for pharmacokinetic study. The analytes and internal standard (IS) were extracted from rat plasma by acetidin. An MS/MS detection was conducted using multiple reaction monitoring (MRM) and operating in the negative ionization mode. The calibration curve ranges were 5–500, 5–500, 2.5–250, 1–100, 1–100, 1–100, and 1–100 ng/ml for BB, GA, GB, GC, QCT, KMF, and ISR, respectively. The mean recovery of the analytes ranged from 68.11% to 84.42%. The intra- and inter-day precisions were in the range of 2.33%–9.86% and the accuracies were between 87.67% and 108.37%. The method was used successfully in a pharmacokinetic study of GBTs. The pharmacokinetic parameters of seven compounds were analyzed using a non-compartment model. Plasma concentrations of the seven compounds were determined up to 48 h after administration, and their pharmacokinetic parameters were in agreement with previous studies.
doi:10.1631/jzus.B1400035
PMCID: PMC4228506  PMID: 25367786
Ginkgo biloba tablet; Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); Pharmacokinetics
20.  Essential oil from Zanthoxylum bungeanum Maxim. and its main components used as transdermal penetration enhancers: a comparative study*  
Our previous studies had confirmed that the essential oil from Zanthoxylum bungeanum Maxim. (Z. bungeanum oil) could effectively enhance the percutaneous permeation of drug molecules as a natural transdermal penetration enhancer. The aim of the present study is to investigate and compare the skin penetration enhancement effect of Z. bungeanum oil and its main components on traditional Chinese medicine (TCM) active components. Toxicities of Z. bungeanum oil and three selected terpene compounds (terpinen-4-ol, 1,8-cineole, and limonene) in epidermal keratinocytes (HaCaT) and dermal fibroblast (CCC-ESF-1) cell lines were measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Five model drugs in TCM external preparations, namely osthole (OT), tetramethylpyrazine (TMP), ferulic acid (FA), puerarin (PR), and geniposide (GP), which were selected based on their lipophilicity denoted by logK o/w, were tested using in vitro permeation studies in which vertical Franz diffusion cells and rat abdominal skin were employed. The secondary structure changes of skin stratum corneum (SC) and drug thermodynamic activities were investigated to understand their mechanisms of action using Fourier transform infrared (FTIR) spectroscopy and saturation solubility studies, respectively. It was found that Z. bungeanum oil showed lower toxicities in both HaCaT cells and CCC-ESF-1 cells compared with three terpene compounds used alone. The enhancement permeation capacities by all tested agents were in the following increasing order: terpinen-4-ol≈1,8-cineole
doi:10.1631/jzus.B1400158
PMCID: PMC4228507  PMID: 25367787
Zanthoxylum bungeanum Maxim.; Essential oil; Limonene; Fourier transform infrared (FTIR) spectroscopy; Penetration enhancer; HaCaT
Ethnicity & disease  2013;23(3):310-315.
Objectives
This research examines the differences in estimated odds of developing diabetes mellitus for white, black, and Mexican-Americans age 51 and over for a period of 11 years.
Design, Setting, and Participants
Longitudinal data came from 14,783 respondents of the Health and Retirement Study (1995–2006) who report being diabetes-free at the first time period. Discrete-time survival models were used to analyze ethnic variations in the probability of developing diabetes.
Main Outcome Measure
Estimated odds of developing diabetes mellitus.
Results
The odds of newly diagnosed diabetes increased between 1995 and 2006, with 11% cumulative incidence for all study participants. The probability of incident diabetes among black Americans was 0.01 during the period of 1995/96–1998, which increased to 0.03 during 1998–2000 and remained at 0.03 throughout subsequent periods, with cumulative incidence over the 11 years at 12%. In contrast, for Mexican-Americans the probability more than doubled from 0.02 in 1995/96–1998 to 0.05 in 2004–2006, with cumulative incidence at 19%. White Americans had 11% cumulative incidence during the 11 year period.
Conclusions
Relative to white Americans, Mexican-Americans had significantly elevated odds of developing diabetes throughout the 11-year period of observation even after controlling for differences in demographic, socioeconomic, and time-varying health characteristics.
PMCID: PMC4106363  PMID: 23914416
Ethnic differences; diabetes mellitus incidence; discrete-time survival analysis
Ethnicity & disease  2012;22(2):175-180.
Objectives
This research examines the differences in estimated risk of developing hypertension in whites, blacks, and Mexican-Americans age 50 and over for a period of 11 years.
Design, Setting, and Participants
Data came from 9,259 respondents who reported being hypertension-free at the baseline in the Health and Retirement Study with up to five time intervals (1998-2006). Discrete-time survival models were used to analyze ethnic variations in the probability of developing hypertension.
Main Outcome Measure
Estimated odds of developing hypertension.
Results
The risk of newly diagnosed hypertension increased between 1995 and 2006 for HRS participants over age 50 years. After adjusting for demographic and health status, the probability of incident hypertension among black Americans was 0.10 during the period of 1995/96-1998, which increased steadily to 0.17 in 2004-2006, and cumulative incidence over the 11-year period at 51%. In contrast, among white Americans the risk was 0.07 during 1995/96-1998 and 0.13 in 2004-2006, with cumulative incidence at 43%. For Mexican-Americans, the probability also increased from 0.08 during 1995/96-1998 to 0.14 during 2004-2006, and cumulative incidence at 42%.
Conclusions
Relative to white and Mexican-Americans, black Americans had an elevated risk of incident hypertension throughout the 11-year period of observation. These variations persisted even when differences in health behaviors, socioeconomic status, demographic, and time-varying health characteristics are accounted for.
PMCID: PMC4084710  PMID: 22764639
Ethnic differences; hypertension incidence; discrete-time survival analysis
PLoS ONE  2014;9(7):e100416.
Background
Natural products present in low quantity in herb medicines constitute an important source of chemical diversity. However, the isolation of sufficient amounts of these low abundant constituents for structural modification has been a challenge for several decades and subsequently halts research on the utilization of this important source of chemical entities for drug discovery and development. And, pro-angiogenic therapies are being explored as options to treat cardio-cerebral vascular diseases and wound healing recently. The present study investigates the pro-angiogenic potential of tanshinone derivatives produced by one-pot synthesis using zebrafish model.
Methodology/Principal Findings
In order to address the difficulty of chemical modification of low abundant constituents in herb medicines, a novel one-pot combinatorial modification was used to diversify a partially purified tanshinone mixture from Salvia miltiorrhiza. This led to the isolation of ten new imidazole-tanshinones (Compounds 1–10) and one oxazole-tanshinone (Compound 11), the structures of which were characterized by spectroscopic methods in combination with single-crystal X-ray crystallographic analysis. The angiogenesis activities of the new tanshinone derivatives were determined in an experimental model of chemical-induced blood vessels damage in zebrafish. Of all the tested new derivatives, compound 10 exhibited the most potent vascular protective and restorative activity with an EC50 value of 0.026 µM. Moreover, the mechanism underlying the pro-angiogenesis effect of 10 probably involved the VEGF/FGF-Src-MAPK and PI3K-P38 signalling pathways by gene expression analysis and a blocking assay with pathways-specific kinase inhibitors.
Conclusions/Significance
Taken together, our study demonstrated the more distinctive pro-angiogenic properties of 10 than other tanshinones and revealed 10 has potential for development as a pro-angiogenic agent for diseases associated with insufficient angiogenesis. Our results highlighted the great potential of adopting a newly modified one-pot approach to enhance the chemical diversity and biological activities of constituents from natural products regardless of their abundances.
doi:10.1371/journal.pone.0100416
PMCID: PMC4081027  PMID: 24992590
Introduction and hypothesis
This study describes pelvic organ support after childbirth.
Methods
This ancillary analysis of the Childbirth and Pelvic Symptoms Imaging Study compares pelvic organ prolapse quantification 6–12 months after childbirth among three cohorts of primiparous women: vaginal delivery with sphincter tear (n=106), vaginal delivery without sphincter tear (n=108), and cesarean without labor (n=39).
Results
Of participants, 31.2% had stage II support. Prolapse to or beyond the hymen was present in 14% after vaginal delivery with sphincter tear (95% confidence interval 8%, 22%), 15% (9%, 24%) after vaginal delivery without sphincter tear, and 5% (1%, 17%) after cesarean without labor (p=0.23). A study of 132 women per group would be required for 80% power to test differences between 5% and 15%.
Conclusions
While these data provide insufficient power to dismiss a difference in pelvic organ support between modes of delivery, they add to our understanding of support following childbirth.
doi:10.1007/s00192-009-0937-3
PMCID: PMC4064938  PMID: 19777148
Pelvic organ prolapse quantification; Uterine prolapse; Childbirth; Cesarean; Obstetrical anal sphincter laceration
Background
The long-term ingestion of alcohol diminishes hypothalamic–pituitary–adrenal (HPA) axis reactivity in alcohol-dependent men, potentially altering future relapse risk. Although sex differences in HPA axis functioning are apparent in healthy controls, disruptions in this system have received little attention in alcohol-dependent women. In this study, we assessed the basal secretory profile of adrenocorticotropic hormone (ACTH) and cortisol, adrenocortical sensitivity in both the presence and absence of endogenous corticotropic pituitary activation, and feedback pituitary glucocorticoid sensitivity to dexamethasone.
Methods
Seven women 4- to 8-week abstinent alcohol-only dependent subjects and 10 age-matched female healthy controls were studied. All subjects were between 30 and 50 years old, not taking oral contraceptives, and were studied during the early follicular phase of their menstrual cycle. Circulating concentrations of ACTH and cortisol were measured in blood samples collected at frequent intervals from 2000 to 0800 hour. A submaximal dose of cosyntropin (0.01 μg/kg), a synthetic ACTH (1–24), was administered at 0800 hour to assess adrenocortical sensitivity. In a separate session, low-dose cosyntropin was also administered following high-dose dexamethasone (8 mg intravenous) to assess adrenocortical sensitivity in the relative absence of endogenous ACTH. In addition, the ACTH response to dexamethasone was measured to determine the pituitary glucocorticoid negative feedback. Sessions were 5 days apart, and blood draws were obtained every 5 to 10 minutes.
Results
Mean concentrations and pulsatile characteristics of ACTH and cortisol over 12 hours were not statistically different between the 2 groups. Healthy controls had a somewhat higher (p < 0.08) net peak, but not net integrated, cortisol response to cosyntropin relative to the alcohol-dependent women. There were no significant group differences in either the ACTH or cortisol response to dexamethasone nor in the net cortisol response to cosyntropin following dexamethasone.
Conclusion
Significant differences in pituitary–adrenal function were not apparent between alcohol-dependent women and matched controls. Despite the small n, it appears that alcohol-dependent women do not show the same disruptions in HPA activity as alcohol-dependent men. These findings may have relevance for gender-specific treatment effectiveness.
doi:10.1111/j.1530-0277.2010.01164.x
PMCID: PMC4038906  PMID: 20331575
Adrenal Cortex; Alcoholism; Cosyntropin; Dexamethasone; Pituitary-Adrenal System; Gender; Female

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