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1.  Electroporation of Functional Bacterial Effectors into Mammalian Cells 
The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.
doi:10.3791/52296
PMCID: PMC4331347  PMID: 25650771
Immunology; Issue 95; electroporation; protein; transfection; expression; localization; confocal microscopy; Salmonella; effector
2.  Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing 
JAMA  2014;312(18):1870-1879.
IMPORTANCE
Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders.
OBJECTIVE
To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome.
DESIGN, SETTING, AND PATIENTS
Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patient’s physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay.
MAIN OUTCOMES AND MEASURES
Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings.
RESULTS
A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%–31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%–27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%–47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%–25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics.
CONCLUSIONS AND RELEVANCE
Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.
doi:10.1001/jama.2014.14601
PMCID: PMC4326249  PMID: 25326635
3.  Finding Our Way through Phenotypes 
Deans, Andrew R. | Lewis, Suzanna E. | Huala, Eva | Anzaldo, Salvatore S. | Ashburner, Michael | Balhoff, James P. | Blackburn, David C. | Blake, Judith A. | Burleigh, J. Gordon | Chanet, Bruno | Cooper, Laurel D. | Courtot, Mélanie | Csösz, Sándor | Cui, Hong | Dahdul, Wasila | Das, Sandip | Dececchi, T. Alexander | Dettai, Agnes | Diogo, Rui | Druzinsky, Robert E. | Dumontier, Michel | Franz, Nico M. | Friedrich, Frank | Gkoutos, George V. | Haendel, Melissa | Harmon, Luke J. | Hayamizu, Terry F. | He, Yongqun | Hines, Heather M. | Ibrahim, Nizar | Jackson, Laura M. | Jaiswal, Pankaj | James-Zorn, Christina | Köhler, Sebastian | Lecointre, Guillaume | Lapp, Hilmar | Lawrence, Carolyn J. | Le Novère, Nicolas | Lundberg, John G. | Macklin, James | Mast, Austin R. | Midford, Peter E. | Mikó, István | Mungall, Christopher J. | Oellrich, Anika | Osumi-Sutherland, David | Parkinson, Helen | Ramírez, Martín J. | Richter, Stefan | Robinson, Peter N. | Ruttenberg, Alan | Schulz, Katja S. | Segerdell, Erik | Seltmann, Katja C. | Sharkey, Michael J. | Smith, Aaron D. | Smith, Barry | Specht, Chelsea D. | Squires, R. Burke | Thacker, Robert W. | Thessen, Anne | Fernandez-Triana, Jose | Vihinen, Mauno | Vize, Peter D. | Vogt, Lars | Wall, Christine E. | Walls, Ramona L. | Westerfeld, Monte | Wharton, Robert A. | Wirkner, Christian S. | Woolley, James B. | Yoder, Matthew J. | Zorn, Aaron M. | Mabee, Paula
PLoS Biology  2015;13(1):e1002033.
Imagine if we could compute across phenotype data as easily as genomic data; this article calls for efforts to realize this vision and discusses the potential benefits.
Despite a large and multifaceted effort to understand the vast landscape of phenotypic data, their current form inhibits productive data analysis. The lack of a community-wide, consensus-based, human- and machine-interpretable language for describing phenotypes and their genomic and environmental contexts is perhaps the most pressing scientific bottleneck to integration across many key fields in biology, including genomics, systems biology, development, medicine, evolution, ecology, and systematics. Here we survey the current phenomics landscape, including data resources and handling, and the progress that has been made to accurately capture relevant data descriptions for phenotypes. We present an example of the kind of integration across domains that computable phenotypes would enable, and we call upon the broader biology community, publishers, and relevant funding agencies to support efforts to surmount today's data barriers and facilitate analytical reproducibility.
doi:10.1371/journal.pbio.1002033
PMCID: PMC4285398  PMID: 25562316
4.  Radon Concentrations in Drinking Water in Beijing City, China and Contribution to Radiation Dose 
222Rn concentrations in drinking water samples from Beijing City, China, were determined based on a simple method for the continuous monitoring of radon using a radon-in-air monitor coupled to an air-water exchanger. A total of 89 water samples were sampled and analyzed for their 222Rn content. The observed radon levels ranged from detection limit up to 49 Bq/L. The calculated arithmetic and geometric means of radon concentrations in all measured samples were equal to 5.87 and 4.63 Bq/L, respectively. The average annual effective dose from ingestion of radon in drinking water was 2.78 μSv, and that of inhalation of water-borne radon was 28.5 μSv. It is concluded that it is not the ingestion of waterborne radon, but inhalation of the radon escaping from water that is a substantial part of the radiological hazard. Radon in water is a big concern for public health, especially for consumers who directly use well water with very high radon concentration.
doi:10.3390/ijerph111111121
PMCID: PMC4245603  PMID: 25350007
radon; drinking water; well water; public water; radiation dose
5.  Etanercept in the treatment of ankylosing spondylitis: A systematic review and meta-analysis 
Etanercept (ETN) has been widely applied in the treatment of ankylosing spondylitis (AS). As the use of ETN has increased, associated adverse effects have been reported frequently. Previous meta-analyses have focused on comparing the differences in clinical outcomes between ETN and placebo (PBO). The present meta-analysis evaluated randomised controlled trials (RCTs) to compare the effects of ETN and a PBO or sulfasalazine (SSZ) in patients with AS. The study population characteristics and the main results, including the Assessment in AS 20% response (ASAS 20), the Bath AS Disease Activity Index (BASDAI) and the Bath AS Functional Index (BASFI), were extracted. The pooled odds ratios (ORs) or weighted mean differences (MDs) were calculated using a fixed or random effects model. Fifteen randomised controlled trials (RCTs) involving 2,194 subjects were included. Compared with a PBO, ETN significantly improved the ASAS 20 [P<0.00001; OR, 8.25; 95% confidence interval (CI), 5.92–11.50], BASDAI (P<0.00001; MD, −18.81; 95% CI, −24.47 to −13.15) and BASFI (P<0.00001; standard MD, −0.68; 95% CI, −0.85 to −0.50). In comparison with SSZ, ETN significantly decreased the BASDAI (P<0.00001; MD, −2.40; 95% CI, −2.89 to −1.90) and C-reactive protein (CRP) levels (P<0.0001; MD, −8.01; 95% CI, −11.73 to −4.29). The most common adverse effect of ETN was an injection site reaction. This meta-analysis shows that ETN monotherapy is effective in improving physical function and reducing disease activity in patients with AS. Compared with SSZ, ETN markedly decreased the BASDAI and CRP levels. However, the efficacy of ETN in treating AS requires further evaluation by more RCTs in a larger population of patients prior to recommending ETN as a substitute for synthetic disease-modifying antirheumatic drug (DMARD) monotherapy, or combinations of synthetic DMARDs.
doi:10.3892/etm.2014.1974
PMCID: PMC4186333  PMID: 25289064
etanercept; ankylosing spondylitis; systematic review; meta-analysis
6.  Nose to tail, roots to shoots: spatial descriptors for phenotypic diversity in the Biological Spatial Ontology 
Background
Spatial terminology is used in anatomy to indicate precise, relative positions of structures in an organism. While these terms are often standardized within specific fields of biology, they can differ dramatically across taxa. Such differences in usage can impair our ability to unambiguously refer to anatomical position when comparing anatomy or phenotypes across species. We developed the Biological Spatial Ontology (BSPO) to standardize the description of spatial and topological relationships across taxa to enable the discovery of comparable phenotypes.
Results
BSPO currently contains 146 classes and 58 relations representing anatomical axes, gradients, regions, planes, sides, and surfaces. These concepts can be used at multiple biological scales and in a diversity of taxa, including plants, animals and fungi. The BSPO is used to provide a source of anatomical location descriptors for logically defining anatomical entity classes in anatomy ontologies. Spatial reasoning is further enhanced in anatomy ontologies by integrating spatial relations such as dorsal_to into class descriptions (e.g., ‘dorsolateral placode’ dorsal_to some ‘epibranchial placode’).
Conclusions
The BSPO is currently used by projects that require standardized anatomical descriptors for phenotype annotation and ontology integration across a diversity of taxa. Anatomical location classes are also useful for describing phenotypic differences, such as morphological variation in position of structures resulting from evolution within and across species.
doi:10.1186/2041-1480-5-34
PMCID: PMC4137724  PMID: 25140222
Anatomy; Spatial relationships; Position; Axes; Reasoning; BSPO; Ontology; Phenotype
7.  Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo 
Cytotechnology  2013;65(4):643-653.
A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.
doi:10.1007/s10616-012-9520-8
PMCID: PMC3720971  PMID: 23475158
Reversine; Aspirin; Cervical carcinoma; Apoptosis
8.  Proteomic analysis of immature rat pups brain in response to hypoxia and ischemia challenge 
Hypoxia and ischemia significantly affects perinatal brain development, even worse in preterm infants. However, the details of the mechanism leading to permanent brain damage after hypoxia-ischemia attack have not been fully elucidated. Proteomics could provide insight into the potential mechanism and help to promote the clinical treatment. In this study, quantitative analysis was performed 24 hours after hypoxia-ischemia using liquid-chromatography mass spectrometry coupled to label-free analysis. Compared to control, 193 proteins were present only in hypoxic-ischemic group. In addition, 34 proteins were more than 2 folds up-regulated and 14 proteins were more than 2 folds down-regulated in hypoxia-ischemia group. Gene Ontology database showed that the majority of differentially expressed proteins comprised mitochondrial proteins et al. Molecular function analysis revealed that the majority of proteins were involved in ion binding et al. Biological process analysis showed that the majority of proteins were involved in response to organic substance et al. STRING 9.0 software analysis were used to explore the complex interactions existed among the proteins. Western blot were used to verify the fold changes of some proteins-microtubule-associated protein 2 and microtubule-associated protein tau. This novel study performed a full-scale screening of the proteomics research in hypoxic-ischemic brain damage of immature rat.
PMCID: PMC4152027  PMID: 25197337
Hypoxia; ischemia; preterm; proteomics
9.  Structural and Functional Characterization of DUF1471 Domains of Salmonella Proteins SrfN, YdgH/SssB, and YahO 
PLoS ONE  2014;9(7):e101787.
Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.
doi:10.1371/journal.pone.0101787
PMCID: PMC4092069  PMID: 25010333
10.  Acupuncture-moxibustion in treating irritable bowel syndrome: How does it work? 
Irritable bowel syndrome (IBS) is a functional intestinal disease characterized by abdominal pain or discomfort and altered bowel habits. It has drawn great attention because of its high prevalence, reoccurring symptoms, and severe influence on patients’ lives. Many clinical studies have demonstrated the efficacy of acupuncture-moxibustion in treating IBS. Increasing attention has been paid to research regarding the action mechanisms of acupuncture-moxibustion for IBS, and the adoption of modern techniques has achieved some progress. This article reviews the latest advances among action mechanism studies from the perspectives of gastrointestinal motility, visceral hypersensitivity, the brain-gut axis, the neuroendocrine system, and the immune system. It is shown that acupuncture-moxibustion can effectively regulate the above items, and thus, this treatment should have a high efficacy in the treatment of IBS. This article also identifies existing problems in current mechanism research and raises several ideas for future studies. Further revelations regarding these action mechanisms will promote the application of acupuncture-moxibustion in treating IBS.
doi:10.3748/wjg.v20.i20.6044
PMCID: PMC4033444  PMID: 24876727
Irritable bowel syndrome; Acupuncture-moxibustion; Mechanism study; Gastrointestinal motility; Visceral hypersensitivity; Brain-gut axis
11.  Association of Saccade Duration and Saccade Acceleration/Deceleration Asymmetry during Visually Guided Saccade in Schizophrenia Patients 
PLoS ONE  2014;9(5):e97308.
Objective
To examine the difference between schizophrenia patients and normal controls on velocity and acceleration of saccade, by using the basic visually guided saccade (VGS) paradigm.
Methods
Eighteen schizophrenia outpatients and fourteen normal controls participated in the VGS task. Multiple indicators, including amplitude, duration, velocity, latency, accuracy rate, acceleration, and deceleration were analyzed. Asymmetric acceleration index (AAI) was introduced to describe the difference between peak acceleration and peak deceleration. The correlation coefficient (RAD) of AAI and duration was computed to examine the difference between schizophrenia patients and normal controls.
Results
No significant difference between patients and normal controls was found on amplitude, duration, latency, and accuracy rate. However, RAD values of schizophrenia patients were significantly lower than the control group.
Conclusion
Compared to normal controls, association of saccade duration and saccade acceleration/deceleration asymmetry during visually guided saccade was lower in schizophrenia patients.
doi:10.1371/journal.pone.0097308
PMCID: PMC4023985  PMID: 24837253
12.  The effect of transcutaneous electrical acupoint stimulation on pregnancy rates in women undergoing in vitro fertilization: a study protocol for a randomized controlled trial 
Trials  2014;15:162.
Background
The latest meta-analysis demonstrated that acupuncture improves pregnancy rates among women undergoing in vitro fertilization-embryo transfer (IVF-ET), and surface acupoint stimulation, such as transcutaneous electrical acupoint stimulation (TEAS), may have the same or better potential.
Methods/Design
To explore the effect of TEAS on the clinical pregnancy rate (CPR) and live birth rate (LBR) compared with real acupuncture and controls in women undergoing IVF, a multicenter, randomized controlled trial will be conducted. The inclusion criteria are the following: infertile women <40 years of age undergoing a fresh IVF or intracytoplasmic sperm injection cycle, and the study will be restricted to women with the potential for a lower success rate as defined by two or more previous unsuccessful ETs (fresh or frozen). Those who have severe illnesses possibly precluding IVF or pregnancy, have FSH levels greater than 20 IU/L, received donor eggs, had been previously randomized for this study or had undergone acupuncture (in any modality) as infertility treatment will be excluded. The subjects will be randomly assigned to the TEAS group (IVF + TEAS), the electro-acupuncture (EA) group (IVF + EA), or the control group (only IVF). A total sample size of 2,220 women is required to detect differences in CPR among the three groups. TEAS or EA treatments will start once every two or three days from day 3 of menstruation in the ovarian stimulation cycle until the day of ET. The parameters of TEAS or EA will be the following: a frequency of 2/100 Hz, a moderate electrical current of 3 to 5 mA for TEAS and 0.8 to 1.0 mA for EA. The primary outcome is CPR. Secondary outcomes are LBR, the number of oocytes aspirated and the total gonadotropin dose used in the stimulation cycle.
Discussion
This study will provide significant evidence for using a new method (TEAS) in IVF.
Trial registration
ClinicalTrials.govID: NCT01608048 (05/24/2012).
doi:10.1186/1745-6215-15-162
PMCID: PMC4020380  PMID: 24886647
acupuncture; transcutaneous electrical acupoint stimulation; TEAS
13.  Alkyl Caffeates Improve the Antioxidant Activity, Antitumor Property and Oxidation Stability of Edible Oil 
PLoS ONE  2014;9(4):e95909.
Caffeic acid (CA) is distributed widely in nature and possesses strong antioxidant activity. However, CA has lower solubility in non-polar media, which limits its application in fat-soluble food. To increase the lipophilicity of natural antioxidant CA, a series of alkyl caffeates were synthesized and their antioxidant and antitumor activities were investigated. The antioxidant parameters, including the induction period, acid value and unsaturated fatty acid content, of the alkyl caffeates in edible oil were firstly investigated. The results indicated that alkyl caffeates had a lower DPPH IC50 (14–23 µM) compared to CA, dibutyl hydroxy toluene (BHT) and Vitamin C (24–51 µM), and significantly inhibited four human cancer cells (SW620, SW480, SGC7901 and HepG2) with inhibition ratio of 71.4–78.0% by a MTT assay. With regard to the induction period and acid value assays, methyl and butyl caffeates had higher abilities than BHT to restrain the oxidation process and improve the stability of edible oil. The addition of ethyl caffeate to oil allowed maintenance of a higher unsaturated fatty acid methyl ester content (68.53%) at high temperatures. Overall, the alkyl caffeats with short chain length (n<5) assessed better oxidative stability than those with long chain length. To date, this is the first report to the correlations among the antioxidant activity, anticancer activity and oxidative stability of alkyl caffeates.
doi:10.1371/journal.pone.0095909
PMCID: PMC3997486  PMID: 24760050
14.  Design of stapled α-helical peptides to specifically activate Wnt/β-catenin signaling 
Cell Research  2013;23(4):581-584.
doi:10.1038/cr.2013.30
PMCID: PMC3616433  PMID: 23439249
15.  Electroconvulsive Therapy on Severe Obsessive-Compulsive Disorder Comorbid Depressive Symptoms 
Psychiatry Investigation  2014;11(2):210-213.
Electroconvulsive therapy (ECT) is not currently used as a first-line treatment for obsessive-compulsive disorder (OCD). However, several related case reports have demonstrated that ECT seems to be effective for severe OCD, especially when first-line therapies have failed. In this study, we describe the courses, detailed parameters, effects, and follow-up information relating to three patients with severe OCD who were treated by modified bifrontal ECT after their first-line anti-OCD treatments pharmacotherapy, behavioral therapy, and cognitive behavioral therapy failed. The number of ECT procedures administered in each case is as follows: Case 1, eight; Case 2, three; and Case 3, four. In all three cases, the patients' depressive symptoms improved considerably after the ECT procedures. In addition, the condition of all three patients' OCD significantly improved and remained stable at regular follow-ups. ECT may play an effective role in treating severe OCD.
doi:10.4306/pi.2014.11.2.210
PMCID: PMC4023099  PMID: 24843380
Obsessive-compulsive disorder; Electroconvulsive therapy; Depression
16.  Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage 
Neural Regeneration Research  2014;9(5):513-518.
Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair. miRNA-9 is involved in the occurrence of many related neurological disorders. Bioinformatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups: control group; oxygen-glucose deprivation group (treatment with 8% O2 + 92% N2 and sugar-free medium for 60 minutes); transfection control group (after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid) and miRNA-9 transfection group (after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid). From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligodendrocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.
doi:10.4103/1673-5374.130077
PMCID: PMC4153509  PMID: 25206848
nerve regeneration; brain injury; miRNA-9; oligodendrocyte lineage gene 1; hypoxic-ischemic; brain damage; premature birth; brain slice culture; NSFC grant; neural regeneration
17.  POU5F1 Enhances the Invasiveness of Cancer Stem-Like Cells in Lung Adenocarcinoma by Upregulation of MMP-2 Expression 
PLoS ONE  2013;8(12):e83373.
Lung cancer is the leading cause of cancer-related human deaths. Exploration of the mechanisms underlying the metastasis of cancer stem-like cells (CSLCs) will open new avenues in lung cancer diagnosis and therapy. Here, we demonstrated that CSLCs-derived from lung adenocarcinoma (LAC) cells displayed highly invasive and migratory capabilities via expressing high levels of POU5F1 and MMP-2. We found that POU5F1 directly regulated MMP-2 transcription via interaction with the promoter of MMP-2. POU5F1 knockdown in LACSLCs reduced MMP-2 protein abundance, leading to inhibition of the cell invasion, migration and tumorigenesis potentials of LAC cells. Clinically, aberrantly high expressions of POU5F1 and MMP-2 were inversely correlated with the survival of LAC patients, and the double-positive POU5F1 and MMP-2 showed the worst prediction for the patient’s poor survival. These results indicate that POU5F1 can bind to the MMP-2 promoter for the degradation of surrounding extracellular matrix, and therefore promote invasive and migratory capabilities of LACSLCs. Moreover, our data implicate that the pathological detection of the double-positive expressions for POU5F1 and MMP-2 will be useful as diagnostic and prognostic biomarkers in LAC to advance anti-metastasis therapy.
doi:10.1371/journal.pone.0083373
PMCID: PMC3875455  PMID: 24386189
18.  Creating Rat Model for Hypoxic Brain Damage in Neonates by Oxygen Deprivation 
PLoS ONE  2013;8(12):e83589.
Current study explores the feasibility of using a non-surgical method of oxygen deprivation to create Hypoxic brain damage in neonatal rats for medical studies. 7-day-old Sprague Dowley (SD) rats were kept in a container with low oxygen level (8%) for 1.5h. A second group had bilateral cephalic artery ligation before the 1.5h-low oxygen treatment, a method similar to the popular Rice method, to expose the brain to both hypoxic and ischemic situations. Short term neural functions and brain water weights were evaluated 1 day after the hypoxic treatment. Brain pathology and histology were also examined at 1 day and 3 days after the hypoxic treatment. Both groups showed impaired neural functions and increased brain water weight compared to the controls. Histology studies also revealed injuries in the subcortex, hippocampus and lateral ventricle in the brains from both groups. There is no significant difference in the degree of brain damages observed in the two groups. Our work demonstrated that oxygen deprivation alone is sufficient to cause brain damages similar to those seen in Hypoxic-ischemic brain disease (HIBD). Because this method avoids the invasive surgical procedure and therefore reduces the stress and mortality of laboratory animals during the experiment, we recommend it to be the favorable method for creating rat models for HIBD studies.
doi:10.1371/journal.pone.0083589
PMCID: PMC3866139  PMID: 24358300
19.  Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides 
Chemistry & Biology  2013;20(11):1386-1398.
Summary
Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.
Graphical Abstract
Highlights
•Maf proteins represent a family of nucleoside triphosphate pyrophosphatases•Maf proteins hydrolyze the canonical nucleotides dTTP, UTP, and CTP•Maf proteins are also active against m5UTP, m5CTP, pseudo-UTP, and m7GTP•Maf structures reveal the molecular mechanisms of their substrate selectivity
Tchigvintsev et al. show that Maf proteins are a family of nucleotide pyrophosphatases active against both canonical and modified nucleotides. This suggests that Mafs might have a dual role in cell division and in the prevention of the incorporation of modified nucleotides into cellular nucleic acids.
doi:10.1016/j.chembiol.2013.09.011
PMCID: PMC3899018  PMID: 24210219
20.  Bone marrow mesenchymal stem cells protect against retinal ganglion cell loss in aged rats with glaucoma 
Glaucoma is a common eye disease in the aged population and has severe consequences. The present study examined the therapeutic effects of bone marrow mesenchymal stem cell (BMSC) transplantation in preventing loss of visual function in aged rats with glaucoma caused by laser-induced ocular hypertension. We found that BMSCs promoted survival of retinal ganglion cells in the transplanted eye as compared with the control eye. Further, in swimming tests guided by visual cues, the rats with a BMSC transplant performed significantly better. We believe that BMSC transplantation therapy is effective in treating aged rats with glaucoma.
doi:10.2147/CIA.S47350
PMCID: PMC3817004  PMID: 24204132
glaucoma; stem cell; transplantation; cell therapy; aging
21.  Electro-acupuncture with different current intensities to treat functional constipation: a study protocol for a randomized controlled trial 
Trials  2013;14:344.
Background
Functional constipation (FC) is highly prevalent in the general population of the world and has a substantial negative impact on the health-related quality of life of individuals. Many clinical trials have indicated that acupuncture is effective in the treatment of FC. However, the sample sizes of these previous studies were too small. Furthermore, there are no reports investigating the relationship between the stimulation parameter and the therapeutic effect. We therefore designed a multicenter randomized controlled trial to address these problems and hopefully provide a more conclusive answer to these questions.
Methods
Participants will be included if they meet all of the following conditions: (1) diagnosed with functional constipation according to the Roman III standard; (2) aged between 18 and 65 years; (3) not taking any drugs that promote gastrointestinal movements at least during the 1 week prior to randomization; (3) willing to sign an informed consent form; (4) willing to return to the study site for their study visits. The participants will be randomly assigned to three groups in a 1:1:1 ratio: high current intensity group, low current intensity group, and mosapride citrate control group. The total study period is 9 weeks for each patient, 1 week for baseline, 4 weeks for treatment, and 4 weeks for follow-up. The primary outcome in this trial is the number of defecating events per week. The secondary outcomes will include the shape and properties of the stool, intensity of defecating difficulty, Patient Assessment of Constipation Quality of Life (PAC-QOL), MOS item Short Form health survey (SF-36), Self-Rating Anxiety Scale (SAS), and Self-Rating Depression Scale (SDS).
Discussion
This study will provide significant evidence for the application of acupuncture in FC and will identify a suitable stimulation parameter for treatment.
Trial registration
ClinicalTrials.gov ID: NCT01274793.
doi:10.1186/1745-6215-14-344
PMCID: PMC3854786  PMID: 24143917
Acupuncture; Functional constipation; Study protocol
22.  Proposal of new classification for postoperative patients with hepatocellular carcinoma based on tumor growth characteristics 
AIM: To propose an appropriate staging system for hepatocellular carcinoma (HCC) classification.
METHODS: Here, 288 in-patients with HCC were studied and divided into three groups: those with expansive growth, invasive growth (including satellite nodules, nodule fusions and direct tumor invasion of adjacent organs), or disseminative growth (including vascular involvement, regional lymph node metastasis and distant metastasis). A survival analysis was performed using a Kaplan-Meier analysis, and prognostic factors for overall survival were determined by the Cox proportional hazards regression model.
RESULTS: The overall survival (OS) of patients with invasive tumor growth was shorter than that of patients with expansive tumor growth (27.796 ± 3.730 and 57.398 ± 4.873 mo, respectively, P < 0.001). No significant difference in survival was observed between patients with vascular involvement and patients with regional lymph node metastasis (21.667 ± 4.773 and 14.619 ± 2.456 mo, respectively, P = 0.801). The OS of patients with distant metastasis (6.417 ± 1.395 mo) was shorter than that of the other groups (P < 0.001). No significant difference in survival was observed between patients with expansive tumor growth and vascular and/or regional lymph node involvement and patients with invasive tumor growth and no vascular and/or lymph node involvement (25.762 ± 7.024, 21.200 ± 7.794 and 39.533 ± 5.840 mo, respectively; P = 0.871, 0.307 and 0.563, respectively).
CONCLUSION: These data led to the proposal of a new staging system: the Expansive-Invasive-Disseminative growth staging classification.
doi:10.3748/wjg.v19.i33.5534
PMCID: PMC3761108  PMID: 24023498
Hepatocellular carcinoma; Lymph node; Metastasis; Invasive growth; Staging system classification
23.  Behavioral and biochemical effects of a formulation of the traditional Chinese medicine, Kai-Xin-San, in fatigued rats 
The present study was designed to evaluate the anti-fatigue activity and the behavioral and biochemical effects of Kai-Xin-San (KXS) extracts on fatigued rats. The rats were randomly divided into six groups: untreated control (UC), running control (RC), RC treated with 13 mg/kg/day modafinil and RC treated with KXS at dosages of 125, 250 and 500 mg/kg/day, respectively. The treatments were administered orally. Anti-fatigue activity was assessed using the treadmill running test and serum biochemical parameters were determined using an autoanalyzer and commercially available kits. Furthermore, the standardization of the KXS extracts was ensured using a high-performance liquid chromatography (HPLC)-fingerprint. The extracts were shown to increase exhaustive running time in the treadmill running test and reverse the fatigue-induced reduction in hepatic/muscle glycogen and testosterone, in addition to reducing the lactate dehydrogenase (LDH), serum urea nitrogen (SUN), blood lactic acid (BLA) and β-endorphin levels in the serum of the fatigued rats. Moreover, the extracts enhanced superoxide dismutase (SOD) activity and decreased the malondialdehyde (MDA) levels in the serum of the fatigued rats. The results of this preliminary study indicated that KXS exhibits anti-fatigue activity. This was reflected in the effects on the biochemical markers for fatigue.
doi:10.3892/etm.2013.1256
PMCID: PMC3797299  PMID: 24137300
anti-fatigue effect; Kai-Xin-San; treadmill running test; traditional Chinese medicinal formula
24.  Next-generation sequencing for disorders of low and high bone mineral density 
Introduction
Osteogenesis imperfecta (OI), Ehlers-Danlos syndrome (EDS), and osteopetrosis (OPT)are collectively common inherited skeletal diseases. Evaluation of subjects with these conditions often includes molecular testing which has important counseling, therapeutic and sometimes legal implications. Since several different genes have been implicated in these conditions, Sanger sequencing of each gene can be a prohibitively expensive and time consuming way to reach a molecular diagnosis.
Methods
In order to circumvent these problems, we have designed and tested a NGS platform that would allow simultaneous sequencing on a single diagnostic platform of different genes implicated in OI, OPT, EDS, and other inherited conditions leading to low or high bone mineral density. We used a liquid-phase probe library that captures 602 exons (~100 kb) of 34 selected genes and have applied it to test clinical samples from patients with bone disorders.
Results
NGS of the captured exons by Illumina HiSeq2000 resulted in an average coverage of over 900X. The platform was successfully validated by identifying mutations in 6 patients with known mutations. Moreover, in 4 patients with OI or OPT without a prior molecular diagnosis, the assay was able to detect the causative mutations.
Conclusions
In conclusion, our NGS panel provides a fast and accurate method to arrive at a molecular diagnosis in most patients with inherited high or low bone mineral density disorders.
doi:10.1007/s00198-013-2290-0
PMCID: PMC3709009  PMID: 23443412
25.  Analysis of phage Mu DNA transposition by whole-genome Escherichia coli tiling arrays reveals a complex relationship to distribution of target selection protein B, transcription and chromosome architectural elements 
Journal of biosciences  2011;36(4):587-601.
Of all known transposable elements, phage Mu exhibits the highest transposition efficiency and the lowest target specificity. In vitro, MuB protein is responsible for target choice. In this work, we provide a comprehensive assessment of the genome-wide distribution of MuB and its relationship to Mu target selection using high-resolution Escherichia coli tiling DNA arrays. We have also assessed how MuB binding and Mu transposition are influenced by chromosome-organizing elements such as AT-rich DNA signatures, or the binding of the nucleoid-associated protein Fis, or processes such as transcription. The results confirm and extend previous biochemical and lower resolution in vivo data. Despite the generally random nature of Mu transposition and MuB binding, there were hot and cold insertion sites and MuB binding sites in the genome, and differences between the hottest and coldest sites were large. The new data also suggest that MuB distribution and subsequent Mu integration is responsive to DNA sequences that contribute to the structural organization of the chromosome.
PMCID: PMC3712764  PMID: 21857106
A-tracts; MuB; Mu DNA transposition; Fis; nucleoid-associated proteins; target site selection

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