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1.  A comparison of ARMS and direct sequencing for EGFR mutation analysis and Tyrosine Kinase Inhibitors treatment prediction in body fluid samples of Non-Small-Cell Lung Cancer patients 
Background
Epidermal growth factor receptor (EGFR) mutation is strongly associated with the therapeutic effect of tyrosine kinase inhibitors (TKIs) in patients with non-small-cell lung cancer (NSCLC). Nevertheless, tumor tissue that needed for mutation analysis is frequently unavailable. Body fluid was considered to be a feasible substitute for the analysis, but arising problems in clinical practice such as relatively lower mutation rate and poor clinical correlation are not yet fully resolved.
Method
In this study, 50 patients (32 pleural fluids and 18 plasmas) with TKIs therapy experience and with direct sequencing results were selected from 220 patients for further analysis. The EGFR mutation status was re-evaluated by Amplification Refractory Mutation System (ARMS), and the clinical outcomes of TKIs were analyzed retrospectively.
Results
As compared with direct sequencing, 16 positive and 23 negative patients were confirmed by ARMS, and the other 11 former negative patients (6 pleural fluids and 5 plasmas) were redefined as positive, with a fairly well clinical outcome (7 PR, 3 SD, and 1 PD). The objective response rate (ORR) of positive patients was significant, 81.3% (direct sequencing) and 72.7% (ARMS) for pleural fluids, and 80% (ARMS) for plasma. Notably, even reclassified by ARMS, the ORR for negative patients was still relatively high, 60% for pleural fluids and 46.2% for plasma.
Conclusions
When using body fluids for EGFR mutation analysis, positive result is consistently a good indicator for TKIs therapy, and the predictive effect was no less than that of tumor tissue, no matter what method was employed. However, even reclassified by ARMS, the correlation between negative results and clinical outcome of TKIs was still unsatisfied. The results indicated that false negative mutation still existed, which may be settled by using method with sensitivity to single DNA molecule or by optimizing the extraction procedure with RNA or CTC to ensure adequate amount of tumor-derived nucleic acid for the test.
doi:10.1186/1756-9966-30-111
PMCID: PMC3287118  PMID: 22142557
Body Fluids; EGFR Mutation; Direct Sequencing; ARMS; TKIs; NSCLC
2.  Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species 
Parasites & Vectors  2015;8:201.
Background
Rumen flukes parasitize the rumen and reticulum of ruminants, causing paramphistomiasis. Over the years, there has been considerable debate as to whether Paramphistomum leydeni and Paramphistomum cervi are the same or distant species.
Methods
In the present study, the complete mitochondrial (mt) genome of P. leydeni was amplified using PCR-based sequencing and compared with that of P. cervi. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) of P. leydeni specimens (n = 6) and P. cervi specimens (n = 8) was amplified and then sequenced. Phylogenetic relationship of the concatenated amino acid sequence data for 12 protein-coding genes of the two rumen flukes and selected members of Trematoda was evaluated using Bayesian inference (BI).
Results
The complete mt genome of P. leydeni was 14,050 bp in size. Significant nucleotide difference between the P. leydeni mt genome and that of P. cervi (14.7%) was observed. For genetic divergence in ITS-2, sequence difference between P. leydeni and P. cervi was 3.1%, while no sequence variation was detected within each of them. Phylogenetic analysis indicated that P. leydeni and P. cervi are closely-related but distinct rumen flukes.
Conclusions
Results of the present study support the proposal that P. leydeni and P. cervi represent two distinct valid species. The mt genome sequences of P. leydeni provide plentiful resources of mitochondrial markers, which can be combined with nuclear markers, for further comparative studies of the biology of P. leydeni and its congeners from China and other countries.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0823-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-015-0823-4
PMCID: PMC4392617  PMID: 25890015
Paramphistomum leydeni; Paramphistomum cervi; Mitochondrial genome; Nuclear ribosomal DNA; Phylogenetic analysis
3.  Mysterious abrupt carbon-14 increase in coral contributed by a comet 
Scientific Reports  2014;4:3728.
A large and sudden increase in radiocarbon (14C) around AD 773 are documented in coral skeletons from the South China Sea. The 14C increased by ~ 15‰ during winter, and remain elevated for more than 4 months, then increased and dropped down within two months, forming a spike of 45‰ high in late spring, followed by two smaller spikes. The 14C anomalies coincide with an historic comet collision with the Earth's atmosphere on 17 January AD 773. Comas are known to have percent-levels of nitrogen by weight, and are exposed to cosmic radiation in space. Hence they may be expected to contain highly elevated 14C/12C ratios, as compared to the Earth's atmosphere. The significant input of 14C by comets may have contributed to the fluctuation of 14C in the atmosphere throughout the Earth's history, which should be considered carefully to better constrain the cosmic ray fluctuation.
doi:10.1038/srep03728
PMCID: PMC3893640  PMID: 24430984
4.  The Use of Integrative Therapies in Patients with Amyotrophic Lateral Sclerosis in Shanghai, China 
Objective. To investigate the current use of integrative therapies (IT) in the treatment of patients with amyotrophic lateral sclerosis (ALS). Methods. A cross-sectional, multicenter clinical epidemiological survey was conducted in 12 hospitals in Shanghai. We investigated the type and frequency of IT use and determined whether the use of IT correlated with demographic, social, or disease-specific characteristics in our patient population. Results. A total of 231 (89.5%) of 258 patients with ALS were eligible for the study and 229 (99% of all) of 231 reported the use of at least one IT for the treatment of ALS. Vitamins and Chinese herb decoctions, Chinese herb compounds, massage therapy, and acupuncture were the 5 most commonly used therapies. There was a strong association between education level, income, and use of IT. A household income of more than 75,000 RMB ($49,995) correlated with multiple IT use, and married patients used IT more often than single individuals. The main reasons for using IT were to treat weakness and fatigue, muscle atrophy, the development of ALS, depression, insomnia, limb pain or numbness, and side effects associated with Riluzole. Conclusion. The use of IT is common in patients with ALS in Shanghai. Vitamins and TCM are the most used additional therapies and the widespread and largely unexamined use of IT for ALS requires more attention.
doi:10.1155/2013/613596
PMCID: PMC3865630  PMID: 24363770
5.  Circulating tumor cells in HER2-positive metastatic breast cancer patients: a valuable prognostic and predictive biomarker 
BMC Cancer  2013;13:202.
Background
This study was initiated to investigate the prognostic significance of circulating tumor cell (CTC) enumeration and the predictive value of CTC HER2 expression for efficient anti-HER2 therapy in HER2-positive metastatic breast cancer (MBC) patients.
Methods
Sixty HER2-positive MBC patients were enrolled in the present study. Before the initiation of systemic treatment, CTCs from 7.5 ml of blood were analyzed using the CellSearch system. The progression-free survival (PFS) of the patients was estimated using Kaplan-Meier survival curves.
Results
CTCs were detected in 45% (27/60) of the patients, who had shorter median PFS than those without CTCs (2.5 vs. 7.5 months, P = 0.0125). Furthermore, referring to the standard HER2 testing that uses immunohistochemistry (IHC), we proposed a CTC HER2-positive criterion, defined as >30% of CTCs over-expressing HER2. Among patients undergoing anti-HER2 therapy, those with HER2-positive CTCs had longer PFS (8.8 vs. 2.5 months, P = 0.002). Among patients with HER2-positive CTCs, the median PFS for those receiving anti-HER2 therapy was significantly longer than those who were not (8.8 vs. 1.5 months, P = 0.001). Notably, up to 52% (14/27) of the HER2-positive patients were CTC HER2-negative, and anti-HER2 therapy did not significantly improve the median PFS in these patients (2.5 vs. 0.9 months, P = 0.499).
Conclusions
Our findings underscore the necessity of a comprehensive CTC analysis, which may provide valuable prognostic and predictive information for optimizing individually tailored therapies in HER2-positive MBC patients. To test this idea, additional large cohort, multi-center and prospective clinical trials are needed.
doi:10.1186/1471-2407-13-202
PMCID: PMC3643882  PMID: 23617715
7.  Role of Macrophage Migration Inhibitory Factor in the Proliferation of Smooth Muscle Cell in Pulmonary Hypertension 
Mediators of Inflammation  2012;2012:840737.
Pulmonary hypertension (PH) contributes to the mortality of patients with lung and heart diseases. However, the underlying mechanism has not been completely elucidated. Accumulating evidence suggests that inflammatory response may be involved in the pathogenesis of PH. Macrophage migration inhibitory factor (MIF) is a critical upstream inflammatory mediator which promotes a broad range of pathophysiological processes. The aim of the study was to investigate the role of MIF in the pulmonary vascular remodeling of hypoxia-induced PH. We found that MIF mRNA and protein expression was increased in the lung tissues from hypoxic pulmonary hypertensive rats. Intensive immunoreactivity for MIF was observed in smooth muscle cells of large pulmonary arteries (PAs), endothelial cells of small PAs, and inflammatory cells of hypoxic lungs. MIF participated in the hypoxia-induced PASMCs proliferation, and it could directly stimulate proliferation of these cells. MIF-induced enhanced growth of PASMCs was attenuated by MEK and JNK inhibitor. Besides, MIF antagonist ISO-1 suppressed the ERK1/2 and JNK phosphorylation induced by MIF. In conclusion, the current finding suggested that MIF may act on the proliferation of PASMCs through the activation of the ERK1/2 and JNK pathways, which contributes to hypoxic pulmonary hypertension.
doi:10.1155/2012/840737
PMCID: PMC3270469  PMID: 22363104
8.  Clathrin-Mediated Endocytosis in Living Host Cells Visualized through Quantum Dot Labeling of Infectious Hematopoietic Necrosis Virus▿† 
Journal of Virology  2011;85(13):6252-6262.
Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.
doi:10.1128/JVI.00109-11
PMCID: PMC3126507  PMID: 21525360
9.  Regulation of G1 Arrest and Apoptosis in Hypoxia by PERK and GCN2-Mediated eIF2α Phosphorylation12 
Neoplasia (New York, N.Y.)  2010;12(1):61-68.
Hypoxia is a common microenvironment in solid tumors and is correlated with tumor progression by regulating cancer cell survival. Recent studies suggest that activation of double-stranded RNA-dependent protein kinase-like endoplasmic reticulum-related kinase (PERK) and phosphorylation of α subunit of eIF2 (eIF2α) confer cell adaptation to hypoxic stress. However, eIF2α is still phosphorylated at a lowered level in PERK knockout cells under hypoxic conditions. The mechanism for eIF2α kinase(s) (eIF2AK)-increased cell survival is not clear. In this report, we provide evidence that another eIF2AK, the amino acid starvation-dependent general control of amino acid biosynthesis kinase (GCN2), is also involved in hypoxia-induced eIF2α phosphorylation. We demonstrate that both GCN2 and PERK mediate the cell adaptation to hypoxic stress. High levels of eIF2α phosphorylation lead to G1 arrest and protect cells from hypoxia-induced apoptosis. Reduced phosphorylation of eIF2α by knocking out either PERK or GCN2 suppresses hypoxia-induced G1 arrest and promotes apoptosis in accompany with activation of p53 signal cascade. However, totally abolishing phosphorylation of eIF2α inhibits G1 arrest without promoting apoptosis. On the basis of our results, we propose that the levels of eIF2α phosphorylation serve as a “switch” in regulation of G1 arrest or apoptosis under hypoxic conditions.
PMCID: PMC2805884  PMID: 20072654
10.  Metabolomics Approach Reveals Integrated Metabolic Network Associated with Serotonin Deficiency 
Scientific Reports  2015;5:11864.
Serotonin is an important neurotransmitter that broadly participates in various biological processes. While serotonin deficiency has been associated with multiple pathological conditions such as depression, schizophrenia, Alzheimer’s disease and Parkinson’s disease, the serotonin-dependent mechanisms remain poorly understood. This study therefore aimed to identify novel biomarkers and metabolic pathways perturbed by serotonin deficiency using metabolomics approach in order to gain new metabolic insights into the serotonin deficiency-related molecular mechanisms. Serotonin deficiency was achieved through pharmacological inhibition of tryptophan hydroxylase (Tph) using p-chlorophenylalanine (pCPA) or genetic knockout of the neuronal specific Tph2 isoform. This dual approach improved specificity for the serotonin deficiency-associated biomarkers while minimizing nonspecific effects of pCPA treatment or Tph2 knockout (Tph2-/-). Non-targeted metabolic profiling and a targeted pCPA dose-response study identified 21 biomarkers in the pCPA-treated mice while 17 metabolites in the Tph2-/- mice were found to be significantly altered compared with the control mice. These newly identified biomarkers were associated with amino acid, energy, purine, lipid and gut microflora metabolisms. Oxidative stress was also found to be significantly increased in the serotonin deficient mice. These new biomarkers and the overall metabolic pathways may provide new understanding for the serotonin deficiency-associated mechanisms under multiple pathological states.
doi:10.1038/srep11864
PMCID: PMC4495385  PMID: 26154191
11.  Hydrogen Sulfide Maintains Mesenchymal Stem Cell Function and Bone Homeostasis via Regulation of Ca2+ Channel Sulfhydration 
Cell stem cell  2014;15(1):66-78.
SUMMARY
Gaseous signaling molecules such as hydrogen sulfide (H2S) are produced endogenously and mediate effects through diverse mechanisms. H2S is one such gasotrasmitter which regulates multiple signaling pathways in mammalian cells, and abnormal H2S metabolism has been linked to defects in bone homeostasis. Here, we demonstrate that bone marrow mesenchymal stem cells (BMMSCs) produce H2S to regulate their self-renewal and osteogenic differentiation, and H2S deficiency results in defects in BMMSC differentiation. H2S deficiency causes aberrant intracellular Ca2+ influx, due to reduced sulfhydration of cysteine residues on multiple Ca2+ TRP channels. This decreased Ca2+ flux downregulates PKC/Erk-mediated Wnt/β-catenin signaling which controls osteogenic differentiation of BMMSCs. Consistently, H2S-deficient mice display an osteoporotic phenotype, which can be rescued by small molecules which release H2S. These results demonstrate H2S regulates BMMSCs, and restoring H2S levels via non-toxic donors may provide treatments for diseases such as osteoporosis which can arise from H2S deficiencies.
doi:10.1016/j.stem.2014.03.005
PMCID: PMC4082757  PMID: 24726192
12.  Baicalein reduces the occurrence of cirrhotic endotoxemia by reducing intestinal mucosal apoptosis 
Background
The dried roots of Scutellaria baicalensis Georgi, is known in traditional Chinese medicine as Huang Qin (H. qin), and it has been officially and traditionally used in treatment of various diseases such as hepatitis in China. Baicalein (BA), a flavonoid originally isolated from H. qin, has shown a wide range of biological activities. This study was to evaluate whether baicalein, can reduce the intestinal mucosal cell apoptosis caused by cirrhotic endotoxemia and its possible mechanisms.
Methods
For this purpose, compound factors modeling was used to establish endotoxemic cirrhotic rat model. Firstly, we evaluated endotoxin, ALT, AST and TBIL levels after the baicalein treatment (20 mg/kg, i.v.). To investigate the mechanism of baicalein effect on apoptosis, TUNEL assay was used to detect intestinal mucosal apoptosis. RT-PCR was used to detect the expression levels of gene Bcl-2 mRNA and Bax mRNA in intestinal mucosal tissues. Caspase-3 activity of intestinal tissue was detected with colorimetric method in our experiments.
Results
After treatment with BA, the serum endotoxin concentration, the intestinal mucosal apoptosis rate and the activity of caspase-3 of the baicalein group were significantly lower than that of the model and the glutamine group. The serum ALT, AST and TBIL concentration of the BA group were significantly lower than that of the model group. The body weight of the baicalein group was significantly lower than that of the normal group, but it was higher than that of the model group. Among the treatment groups, the mRNA level of anti-apoptotic gene Bcl-2 was the lowest in the model group and the highest in the baicalein group while the mRNA level of pro-apoptotic gene Bax was the lowest in the baicalein group and the highest in the model group.
Conclusion
The present results demonstrated that baicalein could reduce the occurrence of cirrhotic endotoxemia partly by reducing intestinal mucosal apoptosis.
doi:10.1186/s12906-015-0682-8
PMCID: PMC4448291  PMID: 26021373
Baicalein; Liver cirrhosis; Endotoxemia; Intestinal mucosa; Apoptosis
13.  Acupuncture attenuates cognitive deficits and increases pyramidal neuron number in hippocampal CA1 area of vascular dementia rats 
Background
Decreased cognition is recognized as one of the most severe and consistent behavioral impairments in dementia. Experimental studies have reported that acupuncture may improve cognitive deficits, relieve vascular dementia (VD) symptoms, and increase cerebral perfusion and electrical activity.
Methods
Multi-infarction dementia was modeled in rats with 3% microemboli saline suspension. Two weeks after acupuncture at Zusanli (ST36), all rats were subjected to a hidden platform trial to test their 3-day spatial memory using the Morris water maze test. To estimate the numbers of pyramidal neuron, astrocytes, and synaptic boutons in hippocampal CA1 area, we adopted an unbiased stereology method to accurately sample and measure the size of cells.
Results
We found that acupuncture at ST36 significantly decreased the escape latency of VD rats. In addition, acupuncture significantly increased the pyramidal neuron number in hippocampal CA1 area (P < 0.05) and tended to decrease the number of astrocytes (P = 0.063). However, there was no significant change in the synaptic bouton number of hippocampal CA1 area in any of the groups (P > 0.05).
Conclusions
These findings suggest that acupuncture may improve cognitive deficits and increase pyramidal neuron number of hippocampal CA1 area in VD rats.
doi:10.1186/s12906-015-0656-x
PMCID: PMC4426171  PMID: 25928206
Vascular dementia; Cognitive function; Acupuncture; Hippocampus; Unbiased stereology
14.  Morphological study of the posterior osseous structures of subaxial cervical spine in a population from northeastern China 
Background
Laminar screws and lateral mass screws have been increasingly used in the treatment of cervical diseases. The purpose of this study is to determine the morphological characteristics of the posterior anatomical structures of the subaxial cervical vertebrae in a northeastern Chinese population.
Methods
Sixty-one consecutive patients underwent cervical spine computed tomography (CT). We analyzed a total of 610 axial images and 61 sagittal images. The following parameters were measured: lamina outer width (LOW), lamina inner width (LIW), lamina axis length (LAL), lamina transverse angle (LTA), lateral mass longitudinal diameter (LMLD), lateral mass transverse diameter (LMTD), sagittal spinous process length (SSPL), axial spinous process length (ASPL), spinal canal transverse diameter (SCTD), spinal canal longitudinal diameter (SCLD), osseous spinal canal area (OSCA), and Pavlov ratio (PR). The participants were classified into male and female groups and developmental canal stenosis (DCS; PR ≤0.75) and non-DCS (NDCS; PR >0.75) groups.
Results
Significant differences were observed among the different vertebral levels for almost all evaluated parameters, except for LTA and OSCA. Statistical differences were found between the right and left sides in all parameters, except for LIW and LOW. All linear parameters, except for SCLD and the angular parameter LTA, significantly differed between the sexes. Significant differences were found between the DCS and NDCS groups in terms of all parameters, except for SCTD.
Conclusions
Various measurements of the posterior structures of subaxial cervical vertebrae differed between the left and right sides, females and males, and the DCS and NDCS groups. Different techniques for lateral mass screw insertion should be used according to different vertebral level. Only C7 laminar may be able to safely accommodate a 2.5-mm translaminar screw. The study data can help doctors to make better surgical decisions and develop more appropriate implants for northeastern Chinese patients.
doi:10.1186/s13018-015-0194-8
PMCID: PMC4409703  PMID: 25896074
Northeastern Chinese; Subaxial cervical spine; Lateral mass; Lamina; Spinous process; Spinal canal
15.  Comparison of two self-assembled macromolecular prodrug micelles with different conjugate positions of SN38 for enhancing antitumor activity 
7-Ethyl-10-hydroxycamptothecin (SN38), an active metabolite of irinotecan (CPT-11), is a remarkably potent antitumor agent. The clinical application of SN38 has been extremely restricted by its insolubility in water. In this study, we successfully synthesized two macromolecular prodrugs of SN38 with different conjugate positions (chitosan-(C10-OH)SN38 and chitosan-(C20-OH)SN38) to improve the water solubility and antitumor activity of SN38. These prodrugs can self-assemble into micelles in aqueous medium. The particle size, morphology, zeta potential, and in vitro drug release of SN38 and its derivatives, as well as their cytotoxicity, pharmacokinetics, and in vivo antitumor activity in a xenograft BALB/c mouse model were studied. In vitro, chitosan-(C10-OH)SN38 (CS-(10s)SN38) and chitosan-(C20-OH) SN38 (CS-(20s)SN38) were 13.3- and 25.9-fold more potent than CPT-11 in the murine colon adenocarcinoma cell line CT26, respectively. The area under the curve (AUC)0–24 of SN38 after intravenously administering CS-(10s)SN38 and CS-(20s)SN38 to Sprague Dawley rats was greatly improved when compared with CPT-11 (both P<0.01). A larger AUC0–24 of CS-(20s)SN38 was observed when compared to CS-(10s)SN38 (P<0.05). Both of the novel self-assembled chitosan-SN38 prodrugs demonstrated superior anticancer activity to CPT-11 in the CT26 xenograft BALB/c mouse model. We have also investigated the differences between these macromolecular prodrug micelles with regards to enhancing the antitumor activity of SN38. CS-(20s)SN38 exhibited better in vivo antitumor activity than CS-(10s)SN38 at a dose of 2.5 mg/kg (P<0.05). In conclusion, both macromolecular prodrug micelles improved the in vivo conversion rate and antitumor activity of SN38, but the prodrug in which C20-OH was conjugated to macromolecular materials could be a more promising platform for SN38 delivery.
doi:10.2147/IJN.S77957
PMCID: PMC4376263  PMID: 25848251
self-assembled prodrug micelles; in vitro cytotoxicity; pharmacokinetics; in vivo antitumor activity
16.  Hypoxia Precondition Promotes Adipose-Derived Mesenchymal Stem Cells Based Repair of Diabetic Erectile Dysfunction via Augmenting Angiogenesis and Neuroprotection 
PLoS ONE  2015;10(3):e0118951.
The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs) for diabetes induced erectile dysfunction (DED). AMSCs were pretreated with normoxia (20% O2, N-AMSCs) or sub-lethal hypoxia (1% O2, H-AMSCs). The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial growth factor (VEGF) and its receptor FIK-1, angiotensin (Ang-1), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg) and were randomly divided into three groups—Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05). Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF) (p<0.01) and smooth muscle markers (α-SMA) (p<0.01). Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.
doi:10.1371/journal.pone.0118951
PMCID: PMC4366267  PMID: 25790284
17.  MiR-25 Protects Cardiomyocytes against Oxidative Damage by Targeting the Mitochondrial Calcium Uniporter 
MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H2O2, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H2O2-induced elevation of mitochondrial Ca2+ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.
doi:10.3390/ijms16035420
PMCID: PMC4394484  PMID: 25764156
cardiomyocytes; miR-25; mitochondrial calcium uniporter; oxidative stress
18.  Application of air insufflation to prevent clinical pancreatic fistula after pancreaticoduodenectomy 
AIM: To introduce an air insufflation procedure and to investigate the effectiveness of air insufflation in preventing pancreatic fistula (PF).
METHODS: From March 2010 to August 2013, a total of 185 patients underwent pancreaticoduodenectomy (PD) at our institution, and 74 patients were not involved in this study for various reasons. The clinical outcomes of 111 patients were retrospectively analyzed. The air insufflation test was performed in 46 patients to investigate the efficacy of the pancreaticojejunal anastomosis during surgery, and 65 patients who did not receive the air insufflation test served as controls. Preoperative assessments and intraoperative outcomes were compared between the 2 groups. Univariate and multivariate analyses were performed to identify the risk factors for PF.
RESULTS: The two patient groups had similar baseline demographics, preoperative assessments, operative factors, pancreatic factors and pathological results. The overall mortality, morbidity, and PF rates were 1.8%, 48.6%, and 26.1%, respectively. No significant differences were observed in either morbidity or mortality between the two groups. The rate of clinical PF (grade B and grade C PF) was significantly lower in the air insufflation test group, compared with the non-air insufflation test group (6.5% vs 23.1%, P = 0.02). Univariate analysis identified the following parameters as risk factors related to clinical PF: estimated blood loss; pancreatic duct diameter ≤ 3 mm; invagination anastomosis technique; and not undergoing air insufflation test. By further analyzing these variables with multivariate logistic regression, estimated blood loss, pancreatic duct diameter ≤ 3 mm and not undergoing air insufflation test were demonstrated to be independent risk factors.
CONCLUSION: Performing an air insufflation test could significantly reduce the occurrence of clinical PF after PD. Not performing an air insufflation test was an independent risk factor for clinical PF.
doi:10.3748/wjg.v21.i6.1872
PMCID: PMC4323465  PMID: 25684954
Pancreatic fistula; Pancreaticoduodenectomy; Air insufflation test; Surgery; Morbidity
19.  Toll-interacting protein (Tollip) negatively regulates pressure overload-induced ventricular hypertrophy in mice 
Cardiovascular Research  2013;101(1):87-96.
Aims
Toll-interacting protein (Tollip) is a critical regulator of the Toll-like receptor-mediated signalling pathway. However, the role of Tollip in chronic pressure overload-induced cardiac hypertrophy remains unclear. This study aimed to determine the functional significance of Tollip in the regulation of aortic banding-induced cardiac remodelling and its underlying mechanisms.
Methods and results
First, we observed that Tollip was down-regulated in human failing hearts and murine hypertrophic hearts, as determined by western blotting and RT–PCR. Using cultured neonatal rat cardiomyocytes, we found that adenovirus vector-mediated overexpression of Tollip limited angiotensin II-induced cell hypertrophy; whereas knockdown of Tollip by shRNA exhibited the opposite effects. We then generated a transgenic (TG) mouse model with cardiac specific-overexpression of Tollip and subjected them to aortic banding (AB) for 8 weeks. When compared with AB-treated wild-type mouse hearts, Tollip-TGs showed a significant attenuation of cardiac hypertrophy, fibrosis, and dysfunction, as measured by echocardiography, immune-staining, and molecular/biochemical analysis. Conversely, a global Tollip-knockout mouse model revealed an aggravated cardiac hypertrophy and accelerated maladaptation to chronic pressure overloading. Mechanistically, we discovered that Tollip interacted with AKT and suppressed its downstream signalling pathway. Pre-activation of AKT in cardiomyocytes largely offset the Tollip-elicited anti-hypertrophic effects.
Conclusion
Our results provide the first evidence that Tollip serves as a negative regulator of pathological cardiac hypertrophy by blocking the AKT signalling pathway.
doi:10.1093/cvr/cvt232
PMCID: PMC3968303  PMID: 24285748
Tollip; Cardiac remodelling; Pressure overload; AKT; Cardiomyocyte hypertrophy
20.  Expression of VEGFR2 and NRP-1 in non-small cell lung cancer and their clinical significance 
Objective
Vascular-targeted therapy is gradually becoming more appealing for patients with lung cancer. It is unclear whether vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) can be biomarkers for clinical treatment. We aimed to investigate the expression levels of VEGFR2 and NRP-1 in human non-small cell lung cancer (NSCLC) and their clinical significance by observing patient prognosis.
Methods
VEGFR2 and NRP-1 were assessed by immunohistochemistry (IHC) in 40 patients with NSCLC and in 10 patients with benign lesions of lung; kinase insert domain receptor (KDR) and NRP-1 copy number gain (CNG) was assessed by fluorescence in situ hybridization (FISH). The distributions of overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method and compared between groups by log-rank test.
Results
Rates of positive immunostaining for VEGFR2 and NRP-1 were 58% and 55%, respectively. KDR and NRP-1 CNG (+) were detected in 32.5% and 30% of tumors, respectively. Levels of both VEGFR2 and NRP-1 in lung tumors were significantly different than in the control tissue (χ2=11.22, P=0.001; χ2=9.82, P=0.001, respectively); similar results were obtained using CNGs (χ2=4.39, P=0.036; χ2=3.95, P=0.046, respectively). Statistically significant correlations were observed with histological grade, clinical TNM stage and the lymph node status (P<0.05), but not age, gender or pathology type (P>0.05). VEGFR2 showed a strong correlation with NRP-1 (Rs=0.68, P=0.00); similar results were observed with KDR and NRP-1 CNG (Rs=0.32, P=0.04). Significant differences in OS and PFS were observed between the groups with higher VEGFR2 and NRP-1 and those with lower expression (P<0.05).
Conclusions
According to these data, VEGFR2 and NRP-1 are highly expressed in NSCLC. We can conclude that they play a key role in NSCLC occurrence, development and metastasis and are associated with patient prognosis (P<0.05 for OS and PFS). This information will be beneficial for clinical anti-angiogenic treatment in NSCLC.
doi:10.3978/j.issn.1000-9604.2014.12.04
PMCID: PMC4279211  PMID: 25561764
Non-small cell lung cancer (NSCLC); vascular endothelial growth factor receptor 2 (VEGFR2); neuropilin-1 (NRP-1); fluorescence in situ hybridization (FISH)
21.  UbcH10 overexpression increases carcinogenesis and blocks ALLN susceptibility in colorectal cancer 
Scientific Reports  2014;4:6910.
Cyclins are essential for cell proliferation, the cell cycle and tumorigenesis in all eukaryotes. UbcH10 regulates the degradation of cyclins in a ubiquitin-dependent manner. Here, we report that UbcH10 is likely involved in tumorigenesis. We found that cancer cells exposed to n-acetyl-leu-leu-norleucinal (ALLN) treatment and UbcH10 depletion exhibit a synergistic therapeutic effect. Abundant expression of UbcH10 drives resistance to ALLN-induced cell death, while cells deficient in UbcH10 were susceptible to ALLN-induced cell death. The depletion of UbcH10 hindered tumorigenesis both in vitro and in vivo, as assessed by colony formation, growth curve, soft agar and xenograft assays. These phenotypes were efficiently rescued through the introduction of recombinant UbcH10. In the UbcH10-deficient cells, alterations in the expression of cyclins led to cell cycle changes and subsequently decreases in tumorigenesis. The tumorigenesis of xenograft tumors from UbcH10-deficient cells treated with ALLN was decreased relative to wild-type cells treated with ALLN in nude mice. On the molecular level, we observed that UbcH10 deficiency enhances the activation of caspase 8 and caspase 3 but not caspase 9 to impair cell viability upon ALLN treatment. Collectively, our results suggest that, as an oncogene, UbcH10 is a potential drug target for the treatment of colorectal cancer.
doi:10.1038/srep06910
PMCID: PMC4223683  PMID: 25376843
22.  Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution 
Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.
doi:10.1038/emi.2014.80
PMCID: PMC4274890  PMID: 26038501
H7N9; influenza virus; neuraminidase; oseltamivir; peramivir
23.  miRNA-124 down-regulates SOX8 expression and suppresses cell proliferation in non-small cell lung cancer 
Non-small lung cell carcinoma (NSCLC) is a leading lethal disease and a global health burden. The function of the Sex determining region Y (SRY)-related high mobility group box (SOX) family gene in cancer has attracted the attention of more and more scientists recently, yet there are few reports regarding the role of SOX in NSCLC. Our study aimed to investigate the expression of SOX8, a protein belonging to the E group of the SOX family, as well as SOX9, in non-small cell lung cancer (NSCLC) and the relationship of gene expression to clinicopathological factors and prognosis in patients. Immunohistochemical analysis was used to measure the expression of SOX8 in 80 NSCLC and 7 adjacent normal tissues. SOX8 expression was detected as elevated in tumor samples and correlated to tumor size (P < 0.001), lymph node metastasis (P = 0.001), differentiation classification (P = 0.015), and clinical stage (P = 0.013) significantly. Moreover, Kaplan-Meier survival analysis demonstrated that shorter survival time for patients who had higher SOX8 expression (P < 0.001). In addition, our experiments indicate that miRNA-124 functions as a tumor suppressor in NSCLC. We also demonstrate miRNA-124 directly targeted and decreased SOX8 in NSCLC cell lines, suggesting smiRNA-124 may regulate NSCLC cell proliferation via decreasing SOX8 (oncogenicity of biomarker in NSCLC).
PMCID: PMC4270566  PMID: 25550787
NSCLC; miRNA-124; Sox8; IHC; lung cancer; survival analysis; cell proliferation
24.  Reduction in dynamin-2 is implicated in ischaemic cardiac arrhythmias 
Ischaemic cardiac arrhythmias cause a large proportion of sudden cardiac deaths worldwide. The ischaemic arrhythmogenesis is primarily because of the dysfunction and adverse remodelling of sarcolemma ion channels. However, the potential regulators of sarcolemma ion channel turnover and function in ischaemic cardiac arrhythmias remains unknown. Our previous studies indicate that dynamin-2 (DNM2), a cardiac membrane-remodelling GTPase, modulates ion channels membrane trafficking in the cardiomyocytes. Here, we have found that DNM2 plays an important role in acute ischaemic arrhythmias. In rat ventricular tissues and primary cardiomyocytes subjected to acute ischaemic stress, the DNM2 protein and transcription levels were markedly down-regulated. This DNM2 reduction was coupled with severe ventricular arrhythmias. Moreover, we identified that the down-regulation of DNM2 within cardiomyocytes increases the action potential amplitude and prolongs the re-polarization duration by depressing the retrograde trafficking of Nav1.5 and Kir2.1 channels. These effects are likely to account for the DNM2 defect-induced arrhythmogenic potentials. These results suggest that DNM2, with its multi-ion channel targeting properties, could be a promising target for novel antiarrhythmic therapies.
doi:10.1111/jcmm.12335
PMCID: PMC4244014  PMID: 25092467
ischaemic cardiac arrhythmias; dynamin-2; ion channels; Nav1.5; Kir2.1
25.  Neuroprotective effects of bilobalide on cerebral ischemia and reperfusion injury are associated with inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation 
Background
Mitogen-activated protein kinase (MAPK) signaling pathways are implicated in inflammatory and apoptotic processes of cerebral ischemia and reperfusion (I/R) injury. Hence, MAPK pathways represent a promising therapeutic target. Exploring the full potential of inhibitors of MAPK pathways is a useful therapeutic strategy for ischemic stroke. Bilobalide, a predominant sesquiterpene trilactone constituent of Ginkgo biloba leaves, has been shown to exert powerful neuroprotective properties, which are closely related to both anti-inflammatory and anti-apoptotic pathways. We investigated the neuroprotective roles of bilobalide in the models of middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reoxygenation (OGD/R) of cerebral I/R injury. Moreover, we attempted to confirm the hypothesis that its protection effect is via modulation of pro-inflammatory mediators and MAPK pathways.
Methods
Male Sprague-Dawley rats were subjected to MCAO for 2 h followed by reperfusion for 24 h. Bilobalide was administered intraperitoneally 60 min before induction of middle cerebral artery occlusion (MCAO). After reperfusion, neurological deficit scores, infarct volume, infarct weight, and brain edema were assessed. Ischemic penumbrae of the cerebral cortex were harvested to determine superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide, TNF-α, interleukin 1β (IL-1β), p-ERK1/2, p-JNK1/2, and p-p38 MAPK concentration. Similarly, the influence of bilobalide on the expression of nitric oxide, TNF-α, IL-1β, p-ERK1/2, p-JNK1/2, and p-p38 MAPK was also observed in an OGD/R in vitro model of I/R injury.
Results
Pretreatment with bilobalide (5, 10 mg/kg) significantly decreased neurological deficit scores, infarct volume, infarct weight, brain edema, and concentrations of MDA, nitric oxide, TNF-α, IL-1β, and increased SOD activity. Furthermore, bilobalide (5, 10 mg/kg) pretreatment significantly down-regulated both p-JNK1/2 and p-p38 MAPK expression, whereas they had no effect on p-ERK1/2 expression in the ischemic penumbra. Supporting these observations in vivo, pretreatment with bilobalide (50, 100 μM) significantly down-regulated nitric oxide, TNF-α, IL-1β, p-JNK1/2, and p-p38 MAPK expression, but did not change p-ERK1/2 expression in rat cortical neurons after OGD/R injury.
Conclusions
These data indicate that the neuroprotective effects of bilobalide on cerebral I/R injury are associated with its inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.
doi:10.1186/s12974-014-0167-6
PMCID: PMC4189683  PMID: 25256700
bilobalide; cerebral ischemia and reperfusion; p-ERK1/2; p-JNK1/2; p-p38 MAPK; pro-inflammatory mediators

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