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2.  Aberrant expression of maternal Plk1 and Dctn3 results in the developmental failure of human in-vivo- and in-vitro-matured oocytes 
Scientific Reports  2015;5:8192.
Fertilisation is the first step in embryonic development, and dynamic changes of key genes may potentially improve assisted reproduction techniques efficiency during this process. Here, we analysed genes that were differentially expressed between oocytes and zygotes and focused on cytokinesis-related genes. Plk1 and Dctn3 were identified as showing dramatic changes in expression during fertilisation and were suggested to play a key role in inducing aneuploidy in zygotes and 8-cell embryos. Moreover, we found that maternal Plk1 and Dctn3 were expressed at lower levels in in vitro matured oocytes, which may have contributed to the high ratio of resulting embryos with abnormal Plk1 and Dctn3 expression levels, thereby reducing the developmental competence of the resulting embryos. Furthermore, the overexpression of Dctn3 can silence Plk1 expression, which suggests a potential regulation mechanism. In conclusion, our present study showed that aberrant expression of Plk1 and Dctn3 increases embryo aneuploidy and developmental failure, particularly in in vitro matured oocytes. Our results facilitate a better understanding of the effects of oocyte maternal gene expression on embryonic development and can be used to improve the outcome of assisted reproduction techniques.
doi:10.1038/srep08192
PMCID: PMC4314639  PMID: 25645239
3.  CD109 Overexpression in Pancreatic Cancer Identified by Cell-Surface Glycoprotein Capture 
Journal of proteomics & bioinformatics  2014;Suppl 10:S10-003-.
Background
The development of novel targeted cancer therapies and/or diagnostic tools is dependent upon an understanding of the differential expression of molecular targets between normal tissues and tumors. Many of these potential targets are cell-surface receptors; however, our knowledge of the cell-surface proteins upregulated in pancreatic tumors is limited, thus impeding the development of targeted therapies for pancreatic cancer. To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, we sought to identify cell-surface proteins that may serve as potential tumor-specfic targets.
Methods
Membrane glycoproteins on the pancreatic cancer cell lines BxPC-3 were labeled with the bifunctional linker biocytin hydrazide. Following proteolytic digestion, biotinylated glycopeptides were captured with streptavidin-coupled beads then released by PNGaseF-mediated endoglycosidase cleavage and identified by liquid chromatography-tandem mass spectrometry (MS). A protein identified by the cell-surface glycoprotein capture procedure, CD109, was evaluated by western analysis of lysates of pancreatic cancer cell lines and by immunohistochemistry in sections of pancreatic ductal adenocarcinoma and non- neoplastic pancreatic tissues.
Results
MS/MS analysis of glycopeptides captured from BxPC-3 cells revealed 18 proteins predicted or known to be associated with the plasma membrane, including CD109, which has not been reported in pancreatic cancer. Western analysis of CD109 in lysates prepared from pancreatic cancer cell lines revealed it was expressed in 6 of 8 cell lines, with a high level of expression in BxPC-3, MIAPaCa-2, and Panc-1 cells. Immunohistochemical analyses of human pancreatic tissues indicate CD109 is significantly overexpressed in pancreatic tumors compared to normal pancreas.
Conclusions
The selective capture of glycopeptides from the surface of pancreatic cancer cell lines can reveal novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas.
doi:10.4172/jpb.S10-003
PMCID: PMC4307595  PMID: 25635161
Pancreatic cancer; Glycoproteins; Proteomic profiling
4.  Loss of heterozygosity 1p/19q and survival in glioma: a meta-analysis 
Neuro-Oncology  2013;16(1):103-112.
Background
Glioma is rarely curable, and factors that influence the prognosis of glioma patients are not fully understood. Loss of heterozygosity (LOH) of 1p/19q has long been known to be a typical molecular signature of oligodendroglial neoplasms. However, whether LOH of 1p/19q is associated with survival in gliomas remains controversial. Here our goal was to evaluate the association between LOH of 1p/19q and progression-free survival (PFS) and overall survival (OS) by conducting a meta-analysis among glioma cases.
Methods
The PubMed and Embase databases were searched from the earliest records to May 2013 to identify studies that met prestated inclusion criteria. Reference lists of retrieved articles were also reviewed. Three authors independently extracted information needed for further analysis. Either a fixed- or a random-effects model was used to calculate the overall combined hazard ratio (HR) estimates.
Results
Twenty-eight eligible studies involving 3 408 cases were included in the meta-analysis. Compared with the chromosomal intact group, codeletion of 1p and 19q was associated with a better PFS (HR = 0.63; 95% CI, 0.52–0.76) and OS (HR = 0.43; 95% CI, 0.35–0.53). Subgroup analyses showed this association to be independent of detection methods and the grades and subtypes of gliomas. Furthermore, isodeletion of chromosome 1p predicted a similar favorable disease outcome (PFS: HR = 0.68; 95% CI, 0.47–0.97) (OS: HR = 0.51; 95% CI, 0.35–0.75), especially in low-grade gliomas, whereas isodeletion of 19q only indicated longer PFS (HR = 0.70; 95% CI, 0.56–0.87).
Conclusion
Codeletion of 1p and 19q is associated with better survival rates in glioma. Isodeletion of 1p predicts similar outcomes but to a lesser extent, whereas the effects of isodeletion of 19q remained only marginal.
doi:10.1093/neuonc/not145
PMCID: PMC3870828  PMID: 24311641
1p; 19q; glioma; meta-analysis; survival
5.  Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure 
World Journal of Gastroenterology : WJG  2014;20(48):18458-18465.
AIM: To study the clinical efficacy of traditional Chinese medicine (TCM) intervention “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) for treating liver failure due to chronic hepatitis B.
METHODS: We designed the study as a randomized controlled clinical trial. Registration number of Chinese Clinical Trial Registry is ChiCTR-TRC-12002961. A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study. Participants were randomly assigned to the following three groups: (1) a modern medicine control group (MMC group, 36 patients); (2) a “tonifying qi and detoxification” (“TQD”) group (72 patients); and (3) a “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) group (36 patients). Patients in the MMC group received general internal medicine treatment; patients in the “TQD” group were given a TCM formula “tonifying qi and detoxification” and general internal medicine treatment; patients in the “TTK” group were given a TCM formula of “TTK” and general internal medicine treatment. All participants were treated for 8 wk and then followed at 48 wk following their final treatment. The primary efficacy end point was the patient fatality rate in each group. Measurements of various virological and biochemical indicators served as secondary endpoints. The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.
RESULTS: At the 48-wk post-treatment time point, the patient fatality rates in the MMC, “TQD”, and “TTK” groups were 51.61%, 35.38%, and 16.67%, respectively, and the differences between groups were statistically significant (P < 0.05). However, there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups (P > 0.05). Patients in the “TTK” group had significantly higher levels of serum total bilirubin compared to MMC subjects (339.40 μmol/L ± 270.09 μmol/L vs 176.13 μmol/L ± 185.70 μmol/L, P = 0.014). Serum albumin levels were significantly increased in both the “TQD” group and “TTK” group as compared with the MMC group (31.30 g/L ± 4.77 g/L, 30.72 g/L ± 2.89 g/L vs 28.57 g/L ± 4.56 g/L, P < 0.05). There were no significant differences in levels of alanine transaminase among the three groups (P > 0.05). Safety data showed that there was one case of stomachache in the “TQD” group and one case of gastrointestinal side effect in the “TTK” group.
CONCLUSION: Treatment with “TTK” improved the survival rates of patients with liver failure due to chronic hepatitis B. Additionally, liver tissue was regenerated and liver function was restored.
doi:10.3748/wjg.v20.i48.18458
PMCID: PMC4277987  PMID: 25561817
Clinical study; “Tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”); Liver regeneration; Treatment with integrated traditional and Western medicine; Chronic hepatitis B-associated liver failure
6.  Molecular Characterization of the Full Muscovy Duck Parvovirus, Isolated in Guangxi, China 
Genome Announcements  2014;2(6):e01249-14.
We report the complete genomic sequence of the full Muscovy duck parvovirus (MDPV) strain, designated GX2011-5, isolated from a Muscovy duck in Guangxi Province, China. The complete genomic sequence was 5,132 bp in length and contained two major open reading frames encoding a 1,844-nucleotide (nt) nonstructural protein and a 2,199-nt capsid protein. Comparison of the complete sequence of GX2011-5 with other published sequences of Muscovy duck parvovirus revealed that this strain exhibited 90.4% to 95.1% sequence homology. This report will advance our understanding of the epidemiology and molecular characteristics of MDPV in the Muscovy duck population in Guangxi, China.
doi:10.1128/genomeA.01249-14
PMCID: PMC4263826  PMID: 25502664
7.  Blue rubber bleb nevus syndrome: A case report and literature review 
World Journal of Gastroenterology : WJG  2014;20(45):17254-17259.
Blue rubber bleb nevus syndrome (BRBNS) is a rare disease characterized by multiple venous malformations and hemangiomas in the skin and visceral organs. The lesions often involve the cutaneous and gastrointestinal systems. Other organs can also be involved, such as the central nervous system, liver, and muscles. The most common symptoms are gastrointestinal bleeding and secondary iron deficiency anemia. The syndrome may also present with severe complications such as rupture, intestinal torsion, and intussusception, and can even cause death. Cutaneous malformations are usually asymptomatic and do not require treatment. The treatment of gastrointestinal lesions is determined by the extent of intestinal involvement and severity of the disease. Most patients respond to supportive therapy, such as iron supplementation and blood transfusion. For more significant hemorrhages or severe complications, surgical resection, endoscopic sclerosis, and laser photocoagulation have been proposed. Here we present a case of BRBNS in a 45-year-old woman involving 16 sites including the scalp, eyelid, orbit, lip, tongue, face, back, upper and lower limbs, buttocks, root of neck, clavicle area, superior mediastinum, glottis, esophagus, colon, and anus, with secondary severe anemia. In addition, we summarize the epidemiology, clinical manifestations, diagnosis, differential diagnosis and therapies of this disease by analyzing all previously reported cases to enhance the awareness of this syndrome.
doi:10.3748/wjg.v20.i45.17254
PMCID: PMC4258599  PMID: 25493043
Anemia; Blue rubber bleb nevus syndrome; Hemangioma; Vascular malformations; Gastrointestinal bleeding
8.  Spontaneous control of HIV-1 viremia in a subject with protective HLA-B plus HLA-C alleles and HLA-C associated single nucleotide polymorphisms 
Introduction
Understanding the mechanisms by which some individuals are able to naturally control HIV-1 infection is an important goal of AIDS research. We here describe the case of an HIV-1+ woman, CASE1, who has spontaneously controlled her viremia for the last 14 of her 20 years of infection.
Methods
CASE1 has been clinically monitored since 1993. Detailed immunological, virological and histological analyses were performed on samples obtained between 2009 and 2011.
Results
As for other Elite Controllers, CASE1 is characterized by low to undetectable levels of plasma HIV-1 RNA, peripheral blood mononuclear cell (PBMC) associated HIV-1 DNA and reduced in vitro susceptibility of target cells to HIV-1 infection. Furthermore, a slow rate of virus evolution was demonstrated in spite the lack of assumption of any antiretroviral agent. CASE1 failed to transmit HIV-1 to either her sexual male partner or to her child born by vaginal delivery. Normal values and ratios of T and B cells were observed, along with normal histology of the intestinal mucosa. Attempts to isolate HIV-1 from her PBMC and gut-derived cells were unsuccessful, despite expression of normal cell surface levels of CD4, CCRC5 and CXCR4. CASE1 did not produce detectable anti-HIV neutralizing antibodies in her serum or genital mucosal fluid although she displayed potent T cell responses against HIV-1 Gag and Nef. CASE1 also possessed multiple genetic polymorphisms, including HLA alleles (B*14, B*57, C*06 and C*08.02) and HLA-C single nucleotide polymorphisms (SNPs, rs9264942 C/C and rs67384697 del/del), that have been previously individually associated with spontaneous control of plasma viremia, maintenance of high CD4+ T cell counts and delayed disease progression.
Conclusions
CASE1 has controlled her HIV-1 viremia below the limit of detection in the absence of antiretroviral therapy for more than 14 years and has not shown any sign of immunologic deterioration or disease progression. Co-expression of multiple protective HLA alleles, HLA-C SNPs and strong T cell responses against HIV-1 proteins are the most likely explanation of this very benign case of spontaneous control of HIV-1 disease progression.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0335-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-014-0335-6
PMCID: PMC4272524  PMID: 25477316
Human Immunodeficiency Virus (HIV); Viremia; Human Leukocyte Antigen (HLA); Single Nucleotide Polymorphisms (SNPs); Elite controller (ELC); Long term nonprogressor (LTNP)
9.  Isolation and epithelial co-culture of mouse renal peritubular endothelial cells 
BMC Cell Biology  2014;15(1):40.
Background
Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.
Results
Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.
Conclusion
In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.
Electronic supplementary material
The online version of this article (doi:10.1186/s12860-014-0040-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12860-014-0040-6
PMCID: PMC4260259  PMID: 25433516
Peritubular endothelial cells; Tubular epithelial cells; CD146; Co-culture; Vascular endothelial growth factor
10.  RNA-Seq Reveals Leaf Cuticular Wax-Related Genes in Welsh Onion 
PLoS ONE  2014;9(11):e113290.
The waxy cuticle plays a very important role in plant resistance to various biotic and abiotic stresses and is an important characteristic of Welsh onions. Two different types of biangan Welsh onions (BG) were selected for this study: BG, a wild-type covered by wax, which forms a continuous lipid membrane on its epidermal cells, and GLBG, a glossy mutant of BG whose epidermal cells are not covered by wax. To elucidate the waxy cuticle-related gene expression changes, we used RNA-Seq to compare these two Welsh onion varieties with distinct differences in cuticular wax. The de novo assembly yielded 42,881 putative unigenes, 25.41% of which are longer than 1,000 bp. Among the high-quality unique sequences, 22,289 (52.0%) had at least one significant match to an existing gene model. A total of 798 genes, representing 1.86% of the total putative unigenes, were differentially expressed between these two Welsh onion varieties. The expression patterns of four important unigenes that are related to waxy cuticle biosynthesis were confirmed by RT-qPCR and COG class annotation, which demonstrated that these genes play an important role in defense mechanisms and lipid transport and metabolism. To our knowledge, this study is the first exploration of the Welsh onion waxy cuticle. These results may help to reveal the molecular mechanisms underlying the waxy cuticle and will be useful for waxy gene cloning, genetics and breeding as well as phylogenetic and evolutionary studies of the Welsh onion.
doi:10.1371/journal.pone.0113290
PMCID: PMC4240658  PMID: 25415343
11.  Characteristics and long-term prognosis of patients with rectal neuroendocrine tumors 
World Journal of Gastroenterology : WJG  2014;20(43):16252-16257.
AIM: To analyze the clinicopathologic characteristics and prognostic factors of rectal neuroendocrine tumors.
METHODS: The records of 48 patients with rectal neuroendocrine tumors who were treated at the Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, from March 2004 to September 2009 were retrospectively reviewed. The clinicopathological data were extracted and analyzed, and patients were followed-up by telephone or follow-up letter to determine their survival status. Follow-up data were available for all 48 patients. Uni- and multivariate Cox regression analyses were performed to determine the prognostic factors significantly associated with overall survival.
RESULTS: The tumors occurred mostly in the middle and lower rectum, and the most prominent symptoms experienced by patients were hematochezia and diarrhea. The median distance between the tumors and the anal edges was 5.0 ± 2.257 cm, and the median diameter of the tumors was 0.8 ± 1.413 cm. The major pathological type was a typical carcinoid tumor, which accounted for 93.8% (45/48) of patients. Tumor-node-metastasis (TNM) stages I, II, III and IV tumors accounted for 78.8%, 3.9%, 9.6% and 7.7% of patients, respectively. The main treatment method, in 72.9% (35/48) of patients, was transanal extended excision. The 1-, 3- and 5-year survival rates of the whole group of patients were 100%, 93.7%, and 91.3%, respectively. Univariate analysis showed that age (P = 0.032), tumor diameter (P < 0.001), histological type (P < 0.001), TNM stage (P < 0.001), and surgical approach (P = 0.002) were all prognostic factors. On multivariate analysis, only the pathological type was shown to be an independent prognostic factor (HR = 2.797, 95%CI: 1.676-4.668, P = 0.004).
CONCLUSION: In patients with rectal neuroendocrine tumors, TNM stage I is the most common stage found, and lymph node or distant metastases are rarely seen. The pathological type of the tumor is an independent prognostic factor.
doi:10.3748/wjg.v20.i43.16252
PMCID: PMC4239514  PMID: 25473180
Rectal neuroendocrine tumors; Clinical characteristics; Prognostic factors
12.  Mid-Term Results of Metal-on-Metal Hip Resurfacing for Treatment of Osteoarthritis Secondary to Developmental Dysplasia of the Hip: A Minimum of 8-Years of Follow-Up 
Background
Metal-on-metal resurfacing arthroplasty is an attractive alternative to conventional total hip arthroplasty in patients with osteoarthritis secondary to developmental dysplasia of the hip (DDH). The purpose of this study was to assess the mid-term clinical outcome and mid-term survivorship of Metal-on-metal resurfacing arthroplasty in patients suffering from osteoarthritis secondary to DDH.
Material/Methods
Between May 2003 and Dec. 2005, 15 operations using ASR™ and 19 using Corin were performed in 29 patients to treat advanced osteoarthritis secondary to DDHs. There were 6 males (20.7%) and 23 females (79.3%), with an average age of 47.2 years (range, 36–64 years). Clinical and radiographic results were observed. All patients were followed up at the 1st, 2nd, 3rd, 6th, and 12th months after surgery and annually thereafter.
Results
The overall survival was 88.2% at a minimum follow-up of 8 years, but the survival was 91.2% after excluding the infections as the cause of component loosening and failure. The mean Harris hip score improved from 48.27±3.13 (range, 14–71) to 89.63±3.42 (range, 65–100) at latest follow-up. The flexion was from 75.14±8.05° to 107.21±9.34. Only 4 failed because of deep infection, femoral neck fracture, and aseptic loosening.
Conclusions
Metal-on-metal resurfacing arthroplasty showed perfect results at a minimum of 8-years of follow-up in our study, and may be a reasonable option for osteoarthritis secondary to developmental dysplasia of the hip (DDH).
doi:10.12659/MSM.890892
PMCID: PMC4247230  PMID: 25410054
Follow-Up Studies; Hip Dislocation, Congenital; Hip Prosthesis; Osteoarthritis, Hip
13.  Exploring accurate Poisson–Boltzmann methods for biomolecular simulations 
Accurate and efficient treatment of electrostatics is a crucial step in computational analyses of biomolecular structures and dynamics. In this study, we have explored a second-order finite-difference numerical method to solve the widely used Poisson–Boltzmann equation for electrostatic analyses of realistic bio-molecules. The so-called immersed interface method was first validated and found to be consistent with the classical weighted harmonic averaging method for a diversified set of test biomolecules. The numerical accuracy and convergence behaviors of the new method were next analyzed in its computation of numerical reaction field grid potentials, energies, and atomic solvation forces. Overall similar convergence behaviors were observed as those by the classical method. Interestingly, the new method was found to deliver more accurate and better-converged grid potentials than the classical method on or nearby the molecular surface, though the numerical advantage of the new method is reduced when grid potentials are extrapolated to the molecular surface. Our exploratory study indicates the need for further improving interpolation/extrapolation schemes in addition to the developments of higher-order numerical methods that have attracted most attention in the field.
doi:10.1016/j.comptc.2013.09.021
PMCID: PMC3891588  PMID: 24443709
Poisson-Boltzmann equation; Finite difference method; Immersed interface method; Continuum solvent models
14.  Emergency treatment of esophageal varix incarceration in the endoscope and ligation device during endoscopic variceal rubber band ligation 
Sclerotherapy and endoscopic esophageal variceal ligation (EVL) are commonly used to treat and prevent variceal bleeding. As of today, there has been no report on an unexpected incarceration of a varix hooked on with the bands from the endoscopic EVL device. We recently experienced this emergency while using the 7-band ring endoscopic EVL device (Boston Scientific Corp., Boston, MA) for prophylaxis of variceal bleeding. In this case, the varix body itself was accidently incarcerated in the crevice of the esophageal endoscope after highly negative pressure of absorption was applied on the endoscope. In this situation, using force to take out the gastroscope was not an option as it would tear the vein and cause massive hemorrhage. We were managed to ligate the varix with rubber bands while releasing the incarceration. We observed that ligation of the varix at the same position using all seven ligation bands resulted in disappearance of the targeted varix. The surrounding esophageal mucosa became smooth after the treatment.
PMCID: PMC4276241  PMID: 25550983
Esophageal variceal hemorrhage; endoscopic esophageal variceal ligation; emergency situation
15.  Experimental Study on Properties of Methane Diffusion of Coal Block under Triaxial Compressive Stress 
The Scientific World Journal  2014;2014:385039.
Taking the standard size coal block samples defined by ISRM as research objects, both properties of methane diffusion of coal block under triaxial compressive stress and characteristic influences caused by methane pressure were systematically studied with thermo-fluid-solid coupling with triaxial servocontrolled seepage equipment of methane-containing coal. The result shows the methane diffusion property of coal block under triaxial compressive stress was shown in four-stage as follow, first is sharply reduce stage, second is hyperbolic reduce stage, third is close to a fixed value stage, fourth stage is 0. There is a special point making the reduced rate of characteristic curve of methane diffusion speed become sharply small; the influences of shape of methane diffusion speed characteristic curve caused by methane pressure are not obvious, which only is shown in numerical size of methane diffusion speed. Test time was extended required by appear of the special point makes the reduce rate of methane diffusion speed become sharply small. The fitting four-phase relation of methane diffusion of coal block under triaxial compressive stress was obtained, and the idea is proposed that influences of the fitting four-phase relation caused by methane pressure were only shown in value of fitting parameters.
doi:10.1155/2014/385039
PMCID: PMC4235243  PMID: 25531000
16.  The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF-β signaling 
Journal of hepatology  2013;59(5):10.1016/j.jhep.2013.06.025.
Background & Aims
Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis.
Methods
The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-β signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression.
Results
Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-β signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-β signaling.
Conclusions
Thus reciprocal regulation between HCV and SNARK promotes TGF-β signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs.
doi:10.1016/j.jhep.2013.06.025
PMCID: PMC3866804  PMID: 23831117
SNARK; NUAK2; HCV; Metformin; Fibrosis; TGF-beta; SMAD; Kinase
17.  DNA Damage Signaling, Impairment of Cell Cycle Progression, and Apoptosis Triggered by 5-Ethynyl-2′-deoxyuridine Incorporated into DNA 
The “click chemistry” approach utilizing 5-ethynyl-2′-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2, likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.
doi:10.1002/cyto.a.22396
PMCID: PMC3846616  PMID: 24115313
click chemistry; DNA strand breaks; p53 activation; Chk2 activation; ATM activation; γH2AX foci; p53BP1 foci; caspase-3 activation; laser scanning cytometry; flow cytometry; confocal microscopy; SBIP methodology
18.  Cryptosporidium andersoni as a novel predominant Cryptosporidium species in outpatients with diarrhea in Jiangsu Province, China 
BMC Infectious Diseases  2014;14(1):555.
Background
Cryptosporidium hominis and C. parvum are usually considered to be the major pathogens responsible for human cryptosporidiosis. However, there have been few studies regarding the molecular epidemiology of Cryptosporidium in human infections in China. Here we investigated Cryptosporidium infection in patients with diarrhea, in Danyang Hospital of Jiangsu Province, China, at the genotype level.
Methods
A total of 232 stool specimens were collected from outpatients with diarrhea in Danyang Hospital of Jiangsu Province, China, from February 2012 to January 2013. Each specimen was stained from direct fecal smears and examined for Cryptosporidium using modified acid fast staining and microscopy. Moreover, genomic DNA of each fecal sample was screened for the presence of Cryptosporidium with nested PCR, which was genotyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA).
Results
The average infection rate of Cryptosporidium was 1.3% (3/232) by microscopy and subjected to PCR amplification of the SSU rRNA gene of Cryptosporidium, with 9.91% (23/232) being positive for Cryptosporidium with a significant peak in autumn. Based on the SSU rRNA gene, two Cryptosporidium spp. were identified, including C. andersoni (n =21) and C. hominis (n =2). Two types of C. andersoni, designated as A370+ and A370- , were found in the SSU rRNA gene in our present study, which was 100% homologous to C. andersoni infections derived from dairy calves and goats, respectively. The clinical questionnaires showed no significant difference in age, gender and frequency of diarrhea, but duration of diarrhea was shorter for C. andersoni than that of C. hominis (mean, 2 vs. 4 days; p <0.01).
Conclusions
C. andersoni is the dominant species in Danyang City of Jiangsu Province. The fact that SSU rRNA sequences of C. andersoni obtained from human stools exhibited 100% homologous to those derived from dairy calves and goats supported that C. andersoni infection might be attributable to animal origin. The difference in the duration of diarrhea of C. andersoni and C. hominis indicated that different Cryptosporidium species might cause different clinical manifestations.
doi:10.1186/s12879-014-0555-7
PMCID: PMC4225042  PMID: 25344387
Cryptosporidium andersoni; Diarrhea; Stool specimens; Predominant species; Prevalence
19.  Risk factors for predicting symptomatic adjacent segment degeneration requiring surgery in patients after posterior lumbar fusion 
Background
Although measures to reduce and treat degenerative changes after fusion are discussed, these are still controversial.
Methods
A retrospective study was conducted on a consecutive series of 3,799 patients who underwent posterior lumbar fusion for degenerative lumbar disease between January 1999 and January 2009. A total of 28 patients with symptomatic adjacent segment degeneration surgery were identified. Another group of 56 matched patients with degenerative lumbar disease without symptomatic adjacent segment degeneration after spinal fusion were marked as the control group. These two groups were compared for demographic distribution and clinical and radiographic data to investigate the predictive factors of symptomatic adjacent segment degeneration surgery by logistic regression.
Results
The overall incidence rate of symptomatic adjacent segment degeneration surgery was 0.74%. Strong risk factors for the development of a symptomatic adjacent segment degeneration requiring surgery were preoperative distance from L1 to S1 sagittal plumb line (p = 0.031), preoperative lumbar lordosis (p = 0.005), and preoperative adjacent disc height (p = 0.003). Mean postoperative lumbar lordosis was smaller (p = 0.000) in symptomatic adjacent segment degeneration surgery (SASDS) group compared with in the control group (33.3° vs. 39.8°). Postoperative adjacent disc height was also significantly lower in the former group compared with the latter group (p = 0.002). Logistic regression analysis showed that body mass index (BMI) (OR: 1.75; p = 0.006), preoperative adjacent disc degeneration (ADD) on MRI (OR: 13.52; p = 0.027), and disc bulge in preoperative CT examination (OR: 390.4; p = 0.000) maintained their significance in predicting likelihood of symptomatic adjacent segment degeneration surgery.
Conclusions
The occurrence of a symptomatic adjacent segment degeneration surgery is most likely multifactorial and is related to BMI, preoperative ADD on MRI, and disc bulge in preoperative CT examination.
doi:10.1186/s13018-014-0097-0
PMCID: PMC4197214  PMID: 25305779
Lumbar; Adjacent segment degeneration; Fusion; Risk factor
20.  Combined Effects of Admission Serum Creatinine Concentration with Age and Gender on the Prognostic Significance of Subjects with Acute ST-Elevation Myocardial Infarction in China 
PLoS ONE  2014;9(10):e108986.
Objective
to explore the impact of admission serum creatinine concentration on the in-hospital mortality and its interaction with age and gender in patients with acute ST-segment elevation myocardial infarction (STEMI) in China.
Methods
1424 acute STEMI patients were enrolled in the study. Anthropometric and laboratory measurements were collected from every patient. A Cox proportional hazards regression model was used to determine the relationships between the admission serum creatinine level (Cr level), age, sex and the in-hospital mortality. A crossover analysis and a stratified analysis were used to determine the combined impact of Cr levels with age and gender.
Results
Female (HR 1.687, 95%CI 1.051∼2.708), elevated Cr level (HR 5.922, 95%CI 3.780∼9,279) and old age (1.692, 95%CI 1.402∼2.403) were associated with a high risk of death respectively. After adjusting for other confounders, the renal dysfunction was still independently associated with a higher risk of death (HR 2.48, 95% CI 1.32∼4.63), while female gender (HR 1.19, 95%CI 0.62∼2.29) and old age (HR 1.77, 95%CI 0.92∼3.37) was not. In addition, crossover analysis revealed synergistic effects between elevated Cr level and female gender (SI = 3.01, SIM = 2.10, AP = 0.55). Stratified analysis showed that the impact of renal dysfunction on in-hospital mortality was more pronounced in patients <60 years old (odds ratios 11.10, 95% CI 3.72 to 33.14) compared with patients 60 to 74 years old (odds ratios 5.18, 95% CI 2.48∼10.83) and patients ≥75years old (odds ratios 3.99, 95% CI 1.89 to 8.42).
Conclusion
Serum Cr concentration on admission was a strong predictor for in-hospital mortality among Chinese acute STEMI patients especially in the young and the female.
doi:10.1371/journal.pone.0108986
PMCID: PMC4193830  PMID: 25303229
21.  Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo 
Aim
Protein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo.
Methods
The effect of MK-2206 on the growth and proliferation of CNE-1, CNE-2, HONE-1, and SUNE-1 cells was assessed by Cell Counting Kit 8 and colony formation assay. Flow cytometry was performed to analyze cell cycle and apoptosis. The effects of MK-2206 on the AKT pathway were analyzed by Western blotting. Autophagy induction was evaluated via electron microscopy and Western blot. To test the effects of MK-2206 in vivo, CNE-2 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with MK-2206 or placebo. Tumors were harvested for immunohistochemical analysis.
Results
In vitro, MK-2206 inhibited the four NPC cell line growths and reduced the sizes of the colonies in a dose-dependent manner. At 72 and 96 hours, the half maximal inhibitory concentration (IC50) values of MK-2206 in CNE-1, CNE-2, and HONE-1 cell lines were 3–5 μM, whereas in SUNE-1, IC50 was less than 1 μM, and MK-2206 induced cell cycle arrest at the G1 phase. However, our study found no evidence of apoptosis. MK-2206 induced autophagy in NPC cells, as evidenced by electron microscopy and Western blot, and inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of downstream phosphorylation through the PRAS40 and S6 pathways seems to be the main mechanism for the MK-2206-induced growth inhibition.
Conclusion
Our preclinical study suggests that MK-2206’s antiproliferative effect may be useful for NPC treatment; however, strategies for reinforcing this effect are needed to maximize clinical benefit.
doi:10.2147/DDDT.S67961
PMCID: PMC4199975  PMID: 25336925
AKT inhibitor; MK-2206; nasopharyngeal carcinoma
22.  A Study on the Effect of Ethanol Extract of Radix Rhapontici on Erythrocyte Immune Function in Rats 
This paper mainly studied the effect of ethanol extract of Radix rhapontici on erythrocyte immune function in SD rats with acute blood stasis. The methods used the effect on erythrocyte immune function. After intragastric administration of suspension of ethanol extract of Radix rhapontici to SD rats for 3 weeks, on the 21st day from intragastric administration, SD rats were made into blood stasis model and bloods were collected to determine the C3b, C3bRR, RFIR, and RFER in erythrocyte immune function. Meanwhile, serum total antioxidant activity (TAA), superoxide dismutase (SOD) activity, and serum malondialdehyde (MDA) level of rats were determined, and experimental results were analysed with analysis of variance and Q test. The results showed that the ethanol extract of Radix rhapontici had a very good effect on enhancement of erythrocyte immune function in SD rats.
PMCID: PMC3847398  PMID: 24311883
Radix rhapontici; immune; TAA; SOD; MDA
23.  Indel and Carryforward Correction (ICC): a new analysis approach for processing 454 pyrosequencing data 
Bioinformatics  2013;29(19):2402-2409.
Motivation: Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (∼1%) in an infected host with high sensitivity and specificity.
Results: We developed a new approach, referred to as Indel and Carryforward Correction (ICC), to cluster sequences without substitutions and locally correct only indel and carryforward sequencing errors within clusters to ensure that no rare variants are lost. ICC performs sequence clustering in the order of (i) homopolymer indel patterns only, (ii) indel patterns only and (iii) carryforward errors only, without the requirement of a distance cutoff value. Overall, ICC removed 93–95% of sequencing errors found in control datasets. On pyrosequencing data from a PCR fragment derived from 15 HIV-1 plasmid clones mixed at various frequencies as low as 0.1%, ICC achieved the highest sensitivity and similar specificity compared with other commonly used error correction and variant calling algorithms.
Availability and implementation: Source code is freely available for download at http://indra.mullins.microbiol.washington.edu/ICC. It is implemented in Perl and supported on Linux, Mac OS X and MS Windows.
Contact: jmullins@uw.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt434
PMCID: PMC3777116  PMID: 23900188
24.  Essential Role of the Linear Ubiquitin Chain Assembly Complex in Lymphoma Revealed by Rare Germline Polymorphisms 
Cancer discovery  2014;4(4):480-493.
Constitutive activation of NF-κB is a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), owing to upstream signals from the B cell receptor (BCR) and MyD88 pathways. The linear polyubiquitin chain assembly complex (LUBAC) attaches linear polyubiquitin chains to IκB kinase γ, a necessary event in some pathways that engage NF-κB. Two germ line polymorphisms affecting the LUBAC subunit RNF31 are rare among healthy individuals (~1%) but enriched in ABC DLBCL (7.8%). These polymorphisms alter RNF31 α helices that mediate binding to the LUBAC subunit RBCK1, thereby increasing RNF31-RBCK1 association, LUBAC enzymatic activity, and NF-κB engagement. In the BCR pathway, LUBAC associates with the CARD11/MALT1/BCL10 adapter complex and is required for ABC DLBCL viability. A stapled RNF31 α-helical peptide based on the ABC DLBCL-associated Q622L polymorphism inhibited RFN31-RBCK1 binding, decreased NF-κB and killed ABC DLBCL cells, credentialing this protein-protein interface as a therapeutic target.
doi:10.1158/2159-8290.CD-13-0915
PMCID: PMC3992927  PMID: 24491438
25.  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

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