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Find video protocols related to scientific articles indexed in Pubmed.
Interactome Map Reveals Phospholipid Scramblase 1 as a Novel Regulator of Hepatitis B Virus X Protein.
J. Proteome Res.
PUBLISHED: 11-04-2014
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HBV X protein plays crucial roles during viral infection and hepatocellular carcinoma (HCC) development through interaction with various host factors. Here, we mapped the interactome of HBx using a yeast two-hybrid screen. Nine human proteins were identified as novel interacting partners of HBx, one of which is phospholipid scramblase 1 (PLSCR1). PLSCR1 is an interferon-inducible protein that mediates antiviral activity against DNA and RNA viruses. However, the molecular mechanisms of PLSCR1 activity against HBV remain unclear. Here, we reported that PLSCR1 promotes HBx degradation by a proteasome- and ubiquitin-dependent mechanism. Furthermore, we found that PLSCR1 inhibits HBx-mediated cell proliferation. After HBV infection, the protein level of PLSCR1 in plasma is elevated, and chronic hepatitis B patients with low plasma levels of PLSCR1 have a high risk of developing HCC. These results suggest that the nuclear trafficking of PLSCR1 mediates the antiviral activity and anticarcinogenesis against HBV by regulating HBx stability.
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[Intervention of NGAL and HO-1 in valve replacement surgery-induced acute kidney injury].
Zhong Nan Da Xue Xue Bao Yi Xue Ban
PUBLISHED: 10-31-2014
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To determine the pathological mechanism and prevent heart-renal syndrome after heart valve replacement surgery.
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Analysis of curricular reform practices at chinese medical schools.
Teach Learn Med
PUBLISHED: 10-16-2014
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A comprehensive search of the literature published between 2001 and 2010 was performed to gain a greater understanding of curricular reform practices at Chinese medical schools.
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Intermittent Hypoxia-Induced Glucose Intolerance is Abolished by Alpha-Adrenergic Blockade or Adrenal Medullectomy.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 10-16-2014
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Background: Obstructive sleep apnea (OSA) causes intermittent hypoxia (IH) during sleep, and is associated with dysregulation of glucose metabolism. We developed a novel model of clinically realistic IH in mice to test the hypothesis that IH causes hyperglycemia, glucose intolerance, and insulin resistance via activation of the sympathetic nervous system (SNS). Methods: Mice were exposed to acute hypoxia of graded severity (21%, 14%, 10%, 7% O2) or to IH of graded frequency (oxygen desaturation index, ODI of 0, 15, 30, or 60, SpO2 nadir 80%) for 30 minutes to measure levels of glucose fatty acids, glycerol, insulin, and lactate. Glucose tolerance tests and insulin tolerance tests were then performed under each hypoxia condition. Next, we examined these outcomes in mice that were administered phentolamine (? adrenergic blockade), propranolol (? adrenergic blockade) or that underwent adrenal medullectomy before IH exposure. In all experiments, mice were maintained in a thermoneutral environment. Results: Sustained and intermittent hypoxia induced hyperglycemia, glucose intolerance, and insulin resistance in a dose dependent fashion. Only severe hypoxia (7% O2) increased lactate, and only frequent IH (ODI-60) increased plasma fatty acids. Phentolamine or adrenal medullectomy both prevented IH-induced hyperglycemia and glucose intolerance. IH inhibited glucose-stimulated insulin secretion, and phentolamine prevented the inhibition. Propranolol had no effect on glucose metabolism but abolished IH-induced lipolysis. IH-induced insulin resistance was not affected by any intervention. Conclusions: Acutely hypoxia causes hyperglycemia, glucose intolerance, and insulin resistance in a dose-dependent manner. During IH, circulating catecholamines act upon ?-adrenoreceptors to cause hyperglycemia and glucose intolerance.
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Efficacy of adefovir dipivoxil combined with a corticosteroid in 38 cases of nephrotic syndrome induced by hepatitis B virus-associated glomerulonephritis.
Ren Fail
PUBLISHED: 09-24-2014
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To investigate the treatment efficacy of adefovir dipivoxil combined with a corticosteroid on hepatitis B virus-associated glomerulonephritis (HBV-GN).
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[Comparison of mesiodistal tooth angulations determined through traditional panoramic radiographs and cone beam CT panoramic images].
Hua Xi Kou Qiang Yi Xue Za Zhi
PUBLISHED: 09-23-2014
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To compare the accuracy of mesiodistal tooth angulation measurements by using traditional panoramic radiographs and cone beam CT (CBCT) panoramic images.
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Algae separation from urban landscape water using a high density microbubble layer enhanced by micro-flocculation.
Water Sci. Technol.
PUBLISHED: 09-17-2014
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Eutrophication of raw water results in outbreaks of algae, which hinders conventional water treatment. In this study, high density microbubble layers combined with micro-flocculation was adopted to remove algae from urban landscape water, and the effects of pressure, hydraulic loading, microbubble layer height and flocculation dosage on the removal efficiency for algae were studied. The greatest removal efficiency for algae, chemical oxygen demand, nitrogen and phosphorus was obtained at 0.42 MPa with hydraulic loading at 5 m/h and a flocculation dosage of 4 mg/L using a microbubble layer with a height of 130 cm. Moreover, the size, clearance distance and concentration of microbubbles were found to be affected by pressure and the height of the microbubble layer. Based on the study, this method was an alternative for algae separation from urban landscape water and water purification.
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Green synthesis of large-scale highly ordered core@shell nanoporous Au@Ag nanorod arrays as sensitive and reproducible 3D SERS substrates.
ACS Appl Mater Interfaces
PUBLISHED: 09-10-2014
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We present a simple green synthetic approach to large-scale and highly ordered arrays of vertical nanoporous Au nanorods, with an ultrathin Ag-layer in situ electrodeposited on each nanoporous Au nanorod (denoted as core@shell nanoporous Au@Ag nanorod). As both the nanopores within each nanorod and the gaps between the neighboring nanorods create three-dimensional (3D) "hot spots" homogeneously distributed throughout the whole substrate, the core@shell nanoporous Au@Ag nanorod arrays were proved to be sensitive and reproducible surface-enhanced Raman scattering (SERS) substrates. Not only universal probe molecules (rhodamine 6G, R6G) but also nonadsorbing molecules (polychlorinated biphenyls, PCBs) have been detected by using the substrates. After mono-6-thio-?-cyclodextrin (HS-?-CD) was modified to efficiently capture more PCBs molecules, the detection limit of PCBs was further reduced to 5.35 × 10(-7) M. As a trial of practical application, R6G and PCBs with different molar ratios in their mixed solutions were identified, and two congeners of PCBs in their mixture could also be distinguished, showing great potentials in real-time simultaneous detection of multiple pollutants.
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Andrographolide prevents high-fat diet-induced obesity in C57BL/6 mice by suppressing the sterol regulatory element-binding protein pathway.
J. Pharmacol. Exp. Ther.
PUBLISHED: 09-09-2014
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Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the expression of genes involved in biosynthesis of cholesterol, fatty acids, and triglycerides. We investigated the effect of the specific SREBP suppressor andrographolide, a natural compound isolated from Andrographis paniculata, on the regulation of SREBP signaling by use of Western blot, reporter gene assay, and quantitative real-time polymerase chain reaction analysis. In addition, the antiobesity effects of andrographolide were evaluated in C57BL/6 mice with high-fat diet (HFD)-induced obesity. Our results showed that andrographolide downregulated the expressions of SREBPs target genes and decreased cellular lipid accumulation in vitro. Further, andrographolide (100 mg/kg per day) attenuated HFD-induced body weight gain and fat accumulation in liver or adipose tissues, and improved serum lipid levels and insulin or glucose sensitivity in HFD-induced obese mice. Andrographolide effectively suppressed the respiratory quotient, energy expenditure, and oxygen consumption, which may have contributed to the decreased body-weight gain of the obese mice fed with a HFD. Consistently, andrographolide regulated SREBP target genes and metabolism-associated genes in liver or brown adipose tissue, which may have directly contributed to the lower lipid levels and enhanced insulin sensitivity. Taken together, our results indicated that andrographolide ameliorated lipid metabolism and improved glucose use in mice with HFD-induced obesity. Andrographolide has potential as a leading compound in the prevention or treatment of obesity and insulin resistance.
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Screening of latent tuberculosis infection by interferon-? release assays in rheumatic patients: a systemic review and meta-analysis.
Clin. Rheumatol.
PUBLISHED: 09-07-2014
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The aim of this study is to assess the diagnostic value of interferon-? release assays (IGRAs) for latent tuberculosis infection (LTBI) in patients with rheumatic disease before receiving biologic agents. MEDLINE and EMBASE databases were used for searching studies concerning the evaluation on the performance of IGRAs [QuantiFERON-TB Gold (QFT-G), QuantiFERON-TB Gold In-Tube (QFT-GIT) and T-SPOT.TB] in rheumatic patients before biological therapy. After assessing the quality of all studies included in the review, we summarized the results in subgroups using forest plots and calculated pooled estimates if applicable. The search identified 11 studies with a total sample size of 1940 individuals. Compared with the tuberculin skin test (TST), the pooled agreements in QFT-G/GIT and T-SPOT.TB were 72 % (95 % confidence interval (CI) 65, 78 %) and 75 % (95 % CI 67, 83 %), respectively. BCG vaccination was positively correlated with positive rates of TST (pooled odds ratio (OR) 1.64, 95 % CI 1.06, 2.53). Compared with TST, IGRAs were better associated with the presentence of one or more tuberculosis (TB) risk factors. Neither steroid nor disease-modifying anti-rheumatic drugs (DMARDs) significantly affect positive IGRA results. In contrast, TST positivity was significantly impacted by the use of steroid (pooled OR 0.45, 95 % CI 0.30, 0.69), but less significantly by the use of DMARDs (pooled OR 0.78, 95 % CI 0.50, 1.21). In conclusion, in rheumatic patients with previous BCG vaccination or currently on steroid therapy, IGRAs would be the better choice to identify LTBI by decreasing the false-positivity and false-negativity rate compared with conventional TST.
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[A cohort study on the impacts of pre-pregnancy maternal body mass index, gestational weight gain on neonate birth status and perinatal outcomes in Fujian province].
Zhonghua Liu Xing Bing Xue Za Zhi
PUBLISHED: 09-02-2014
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To study the impacts of pre-pregnancy maternal BMI and gestational weight gain(GWG) on pregnancy outcomes.
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Predictive factors for clinical response to allergy immunotherapy in children with asthma and rhinitis.
Int. Arch. Allergy Immunol.
PUBLISHED: 08-16-2014
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To date, no predictive methods for the clinical response to allergy immunotherapy (AIT) are available. We sought to evaluate the pretreatment parameters used in diagnosing allergic asthma and/or rhinitis including allergen skin-prick test responses, serum specific and total IgE (sIgE and tIgE) levels and blood eosinophil counts, and to identify whether these can be used to predict clinical effectiveness in children treated with immunotherapy.
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Carotid body denervation prevents fasting hyperglycemia during chronic intermittent hypoxia.
J. Appl. Physiol.
PUBLISHED: 08-07-2014
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Obstructive sleep apnea causes chronic intermittent hypoxia (IH) and is associated with impaired glucose metabolism, but mechanisms are unknown. Carotid bodies orchestrate physiological responses to hypoxemia by activating the sympathetic nervous system. Therefore, we hypothesized that carotid body denervation would abolish glucose intolerance and insulin resistance induced by chronic IH. Male C57BL/6J mice underwent carotid sinus nerve dissection (CSND) or sham surgery and then were exposed to IH or intermittent air (IA) for 4 or 6 wk. Hypoxia was administered by decreasing a fraction of inspired oxygen from 20.9% to 6.5% once per minute, during the 12-h light phase (9 a.m.-9 p.m.). As expected, denervated mice exhibited blunted hypoxic ventilatory responses. In sham-operated mice, IH increased fasting blood glucose, baseline hepatic glucose output (HGO), and expression of a rate-liming hepatic enzyme of gluconeogenesis phosphoenolpyruvate carboxykinase (PEPCK), whereas the whole body glucose flux during hyperinsulinemic euglycemic clamp was not changed. IH did not affect glucose tolerance after adjustment for fasting hyperglycemia in the intraperitoneal glucose tolerance test. CSND prevented IH-induced fasting hyperglycemia and increases in baseline HGO and liver PEPCK expression. CSND trended to augment the insulin-stimulated glucose flux and enhanced liver Akt phosphorylation at both hypoxic and normoxic conditions. IH increased serum epinephrine levels and liver sympathetic innervation, and both increases were abolished by CSND. We conclude that chronic IH induces fasting hyperglycemia increasing baseline HGO via the CSN sympathetic output from carotid body chemoreceptors, but does not significantly impair whole body insulin sensitivity.
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Linezolid manifests a rapid and dramatic therapeutic effect for patients with life-threatening tuberculous meningitis.
Antimicrob. Agents Chemother.
PUBLISHED: 08-04-2014
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We conducted a retrospective cohort study of patients with MRC grade II/III tuberculous meningitis (TBM) who accepted a background antitubercular regimen (BR) with or without linezolid (LZD). At the 4th week, the LZD-BR group achieved a faster and higher percentage of Glasgow coma scale recovery and temperature recovery, a higher cerebrospinal fluid (CSF)/blood glucose ratio, and lower CSF white blood cell counts than did the BR group. Short-term linezolid supplementation may be a more effective treatment for life-threatening TBM.
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Design of ratiometric emission probe with visible light excitation for determination of Ca2+ in living cells.
Anal. Chem.
PUBLISHED: 08-01-2014
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An organic salt as a fluorescent probe based on intramolecular charge transfer for Ca(2+) determination is developed. Ca(2+) can be detected by ratiometric emission at 490 and 594 nm with an excitation wavelength of 405 nm. This probe is highly selective for Ca(2+) over other divalent metal cations and displays a large Stokes shift of 189 nm that can avoid interference of the excitation light beam and autofluorescence of biological samples. The dissociation constant for Ca(2+) is 2.25 ± 0.47 ?M and pertinent to Ca(2+) detection in cellular resting and dynamic states. The probe demonstrates its application in monitoring Ca(2+) in living cells under confocal microscopic imaging.
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Solid-phase extraction based on chloromethylated polystyrene magnetic nanospheres followed by gas chromatography with mass spectrometry to determine phthalate esters in beverages.
J Sep Sci
PUBLISHED: 07-28-2014
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An ultrasound-assisted magnetic solid-phase extraction procedure with chloromethylated polystyrene-coated Fe3 O4 nanospheres as magnetic adsorbents has been developed to determine eight phthalate esters (bis(4-methyl-2-pentyl) phthalate, dipentyl phthalate, dihexyl phthalate, benzyl butyl phthalate, bis(2-butoxyethyl) phthalate, dicyclohexyl phthalate, di-n-octyl phthalate and dinonyl phthalate) simultaneously in beverage samples, in combination with gas chromatography coupled to tandem mass spectrometry for the first time. Several factors related to magnetic solid-phase extraction efficiencies, such as amount of adsorbent, extracting time, ionic strength and desorption conditions were investigated. The enrichment factors of the method for the eight analytes were over 2482. A good linearity was observed in the range of 10-500 ng/L for bis(2-butoxyethyl) phthalate and 2-500 ng/L for the other phthalate esters with correlation coefficients ranging from 0.9980 to 0.9998. The limits of detection and quantification for the eight phthalate esters were in the range of 0.20-2.90 and 0.67-9.67 ng/L, respectively. The mean recoveries at three spiked levels were 75.8-114.8%, the coefficients of variations were less than 11.6%. The proposed method was demonstrated to be a simple and efficient technique for the trace analysis of the phthalate esters in beverage samples. This article is protected by copyright. All rights reserved.
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Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA.
Vet. J.
PUBLISHED: 07-24-2014
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An indirect porcine epidemic diarrhea virus (PEDV) anti-immunoglobulin (Ig) G ELISA based on the S1 portion of the spike protein was validated and compared with an indirect immunofluorescence assay. In serum samples from experimentally infected pigs (n?=?35), anti-IgG PEDV antibodies were detected as early as 7 days post-infection. In field serum samples (n?=?239), the diagnostic sensitivity of the S1 ELISA was 100% and the diagnostic specificity was 94%. The S1 ELISA showed no cross-reactivity with antibodies against other porcine coronaviruses. Colostrum samples (n?=?133) were also tested for anti-PEDV IgG and IgA. The diagnostic sensitivity was 92% for IgG and 100% for IgA, and the diagnostic specificity was 90% for IgG and 99.4% for IgA. These data suggest that the S1 ELISA is a sensitive and specific test that could also be used to evaluate PEDV colostral immunity.
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[Effects of amantadine and biphenyl dimethyl dicarboxylate on hepatitis B virus in hepatitis B virus replication mice].
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
PUBLISHED: 07-22-2014
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This study sought to investigate the in vivo antiviral effect of amantadine (AM) and biphenyl dimethyl dicarboxylate (DDB) on hepatitis B virus (HBV) in HBV replication mice. HBV replication-competent plasmid was transferred into male BALB/c mice by using hydrodynamics-based in vivo transfection procedure to develop HBV replication mouse model. The model mice were matched by body weigh, age and serum levels of hepatitis B e antigen (HBeAg) and were divided into four groups: AM group, DDB group, AM+DDB group and NS group, with the last one as control, and the mice of each group were administered corresponding agent orally twice a day, in a medication course lasting 3 d. On the third day, the mice were sacrificed 4-6 h after the last oral intake. HBV DNA replication intermediates in liver were analyzed by Southern blot hybridization. The serum hepatitis B surface antigen (HBsAg) and HBeAg were detected by enzyme linked immunosorbent assay (ELISA). Compared to the animals in the control group, HBV DNA replication intermediates in liver and HBsAg and HBeAg in serum from the AM and AM plus DDB group of mice decreased, and there was no difference between these two groups of mice. The levels of HBV DNA intermediate from liver and the serum HBsAg and HBeAg between the control and DDB group, however, were not obviously different. In conclusion, the inhibition effect of AM on HBV was detected, but treatment with DDB for 3 days did not influence the viral replication and expression of HBV in the HBV replication mice.
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Selection of reference genes for normalization of quantitative polymerase chain reaction data in mouse models of heart failure.
Mol Med Rep
PUBLISHED: 07-14-2014
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The accurate measurement of mRNA expression levels is crucially dependent on the use of relevant reference genes for the normalization of data. Currently, heart failure is a serious and widespread disease, and multiple mouse models are utilized for the study of this complex disease. Although mouse models are commonly used to study cardiovascular disease, various studies have not employed the appropriate selection strategies. The present study investigated the expression stability of eight candidate reference genes (GAPDH, ACTB, B2M, CycA, TBP, PBGD, HTRP 1 and 18S) in two mouse models of heart failure, including the transverse aortic arch constriction (TAC) model and the myocardial infarction (MI) model, using GeNorm software. The expression of BNP was normalized using different reference gene strategies, and it was demonstrated that its induction following heart failure was most profound with the optimal reference gene combination. The most stable genes were identified as follows: TBP and CycA in the MI model, and PBGD and GAPDH in the TAC model. The present study provides important information for reference gene selection in mouse models of heart failure, and will aid further investigations of the transcriptome in cardiovascular research.
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Effects of exogenous nesfatin-1 on gastric distention-sensitive neurons in the central nucleus of the amygdala and gastric motility in rats.
Neurosci. Lett.
PUBLISHED: 07-14-2014
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Nesfatin-1 is a novel brain-gut peptide identified in several brain regions associated with feeding and gastrointestinal function. Our study explored the effects of nesfatin-1 in the central nucleus of the amygdala (CNA) on the activity of gastric distention (GD)-sensitive neurons, gastric motility, and the potential regulation mechanisms by the dorsal motor nucleus of the vagus (DMV). Following retrograde injection of fluorogold (FG) into the DMV, we found that nesfatin-1/FG dual-labeled neurons were detected in the CNA, which indicates that some of the nesfatin-1-immunoreactive neurons arising from the DMV may project to the CNA. Single unit discharges in the CNA were recorded extracellularly, and gastric motility was monitored by implantation of a force transducer into the stomach of conscious rats. These results showed that nesfatin-1 administration to the CNA excited most of the GD-excitatory neurons, inhibited GD-inhibitory neurons, and dose-dependently reduced gastric motility. All of the above effects induced by nesfatin-1 could be partially blocked by pretreatment with the melanocortin 3/4 receptors antagonist, SHU9119. Electrical stimulation of the DMV excited the majority of the nesfatin-1-responsive GD neurons in the CNA. Additionally, pretreatment with an anti-NUCB2/nesfatin-1 antibody in the CNA increased the firing rate of nesfatin-1-responsive GD-inhibitory neurons but decreased the firing rate in nesfatin-1-responsive GD-excitatory neurons following electrical stimulation of the DMV. Finally, a subdiaphragmatic vagotomy eliminated the diminished gastric motility induced by nesfatin-1 injection. Taken together, these findings suggest that nesfatin-1 regulates the activity of GD-sensitive neurons and gastric motility via the melanocortin pathway in the CNA. Furthermore, the DMV may be involved in this regulatory pathway.
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Photoluminescent supramolecular hyperbranched polymer without conventional chromophores based on inclusion complexation.
Chem. Commun. (Camb.)
PUBLISHED: 07-12-2014
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A novel photoluminescent supramolecular hyperbranched polymer (SHP) without conventional chromophores was constructed for the first time by inclusion complexation between ?-cyclodextrin and diethylenetriamine. The SHP showed wide-band fluorescence dependent upon the excitation wavelength.
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Placental gene-expression profiles of intrahepatic cholestasis of pregnancy reveal involvement of multiple molecular pathways in blood vessel formation and inflammation.
BMC Med Genomics
PUBLISHED: 07-03-2014
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Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-associated liver disease with potentially deleterious consequences for the fetus, particularly when maternal serum bile-acid concentration >40 ?M. However, the etiology and pathogenesis of ICP remain elusive. To reveal the underlying molecular mechanisms for the association of maternal serum bile-acid level and fetal outcome in ICP patients, DNA microarray was applied to characterize the whole-genome expression profiles of placentas from healthy women and women diagnosed with ICP.
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Emerging salivary biomarkers by mass spectrometry.
Clin. Chim. Acta
PUBLISHED: 06-23-2014
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Human saliva, a multi-constituent oral fluid, has a high potential for early diagnosis of disease. Proteomic analysis of saliva holds promise as a non-invasive method that is advantageous over serum. This non-invasive diagnostic method represents developing trends in analytical and clinical chemistry. Significant technological advances in the field of proteomics during the last two decades have greatly facilitated the research toward this direction. However, these technologies still require integration and standardization of validation against accepted clinical and pathologic parameters. In this review, a summary of mass spectrometry-based technologies of saliva biomarker discovery, potential clinical applications, and challenges of saliva proteomics have been discussed, as well as latest technologies of validation and quantification of saliva biomarkers. It is likely that the use of saliva for early diagnostics of diseases will continue to expand thus providing a new approach of instrumental investigation for physiologic and physiological states. These novel biomarkers have obvious clinical utility that will help to diagnose many diseases at early stage.
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Apoptotic events induced by high glucose in human hepatoma HepG2 cells involve endoplasmic reticulum stress and MAPK's activation.
Mol. Cell. Biochem.
PUBLISHED: 06-18-2014
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To investigate whether endoplasmic reticulum (ER) stress participates in the induction of apoptosis in HepG2 cells exposed to high glucose and explore its probable mechanism. A series of experiments were performed following HepG2 cells treated with different concentrations of glucose for 48 h. The apoptosis was detected by means of Hoechst staining and flow cytometry. Caspase-3 activity assay was performed by measuring the pNA (p-nitroaniline) to indirectly reveal the catalytic activity of caspase-3. The expression levels of apoptosis-, ER stress-associated proteins and MAPKs were analyzed by western blot. To further characterize the molecular mechanisms, the effects of antioxidant alpha-lipoic acid (ALA) and specific inhibitors for JNK and p38 (SP600125 and SB203580, respectively) were examined by Hoechst staining, immunofluorescence, and western blot. After HepG2 cells were incubated with high glucose for 48 h, both Hoechst staining and flow cytometry analyses unveiled the apoptosis of HepG2 cells. Caspase-3 activity assay revealed that the activity of caspase-3 was enhanced. Western blot showed an enhancement of pro-caspase-9 degradation, a reduction of Bcl-2/Bax ratio, a decrease in GRP78 expression, and increases in CHOP and p47/phox levels. In addition, western blot analysis presented that phosphorylation of p38 and JNK was triggered and that the expression of ASK1 was elevated. In the case of the contributions of oxidative stress and the MAPK signaling pathways, all ALA, SP600125 and SB203580 were able to largely rescue high glucose-induced apoptosis. High glucose induced the apoptosis in HepG2 cells through the activation of ASK1-p38/JNK pathway mediated by ER stress and oxidative stress.
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Doppler perfusion index and contrast-enhanced ultrasound in patients with colorectal cancer liver metastases.
Hepatogastroenterology
PUBLISHED: 06-05-2014
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To assess the value of the Doppler perfusion index (DPI) and contrast agent for the detection of liver metastases in patients with colorectal cancer.
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Curriculum reform at Chinese medical schools: What have we learned?
Med Teach
PUBLISHED: 06-04-2014
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Abstract Introduction: Curriculum reform at Chinese medical schools has attracted a lot of attention recently. Several leading medical schools in China have undergone exploratory reforms and in so doing, have accumulated significant experience and have made considerable progress.
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[Genetic analysis and gene mapping of two novel quail-like mutants from the silkworm (Bombyx mori)].
Yi Chuan
PUBLISHED: 05-22-2014
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Two novel body marking mutants were discovered during silkworm (Bombyx mori) breeding. The mutants have no obvious eye-spots compared with normal marking (+) individuals, but their star spots and semilunar markings on dorsal sides are normal, and there are dots and lines with longitudinal wave markings on dorsal sides of the 6th to 7th abdominal segments which consist quail markings in between star spots and semilunar markings. The whole body markings are very similar to that of quail mutant (q); thus these mutants are named as quail-like mutants (q-l). Young larvae of one mutant are in brown color, and develop normally. Their cocoons are regular and uniform in size. Thus, this mutant is designated as brown quail-like (q-lb). Another mutant's larvae are in light purple skin; thus this mutant is named as purple quail-like (q-lp). They take little amount of mulberry leaves, and are weak and develop slowly and unevenly. Their larval bodies and cocoons are small. Genetic analysis revealed that both q-lb and q-lp were recessive genes, and they were allelic, with q-lb recessive to q-lp. These genes are different from quail mutant (q) and located on the chromosome 8 after tested by the morphological markers, P3(2), p(2), Ze(3), L(4), re(5), E(6), q(7), I-a(9), ms(12), ch(13), oa(14), cts(16), mln(18), msn(19), rb(21) and so(26) and SSR markers.
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A snapshot of the status of problem-based learning (PBL) in Chinese medical schools.
Med Teach
PUBLISHED: 05-02-2014
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In North America, where it was born, problem-based learning (PBL) has seen dips and rises in its popularity, but its inherent strengths have led to its spread to medical schools all over the world. Although its use at medical schools in some Western countries has already been examined, no one has looked at its status in many other countries, including China. The aim of this study is to determine the number of schools currently using PBL in China, the degree to which they use it, and the reasoning behind such usage.
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A novel monoclonal antibody against the constant region of goose immunoglobulin light chain.
Monoclon Antib Immunodiagn Immunother
PUBLISHED: 04-22-2014
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A monoclonal antibody (MAb) against the antigenic determinant of the constant region of goose immunoglobulin light chain (GoIgCL) was produced and characterized for the first time here. Goose immunoglobulin (Ig) in serum was purified by immunoaffinity chromatography and the resulting protein was used as immunogen to immunize BALB/c mice. At the same time, the GoIgCL gene was expressed and purified as the screening antigen for selecting MAb against GoIgCL. One hybridoma that produces antibodies against GoIgCL was selected by indirect ELISA. Then the characterization of the MAb was analyzed by ELISA, Western blot, and flow cytometry. It was found to be IgG1 with ? light chain; the MAB has high specificity to Ig in goose serum, bile, and B lymphocytes from peripheral blood, reacts only with the light chain of goose Ig, and can distinguish Ig from other birds. Therefore, the MAb generated in this study can be used as a specific reagent for detection of goose disease-specific antibodies and as a powerful tool for basic immunology research on geese.
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Enhanced cold field emission of large-area arrays of vertically aligned ZnO-nanotapers via sharpening: experiment and theory.
Sci Rep
PUBLISHED: 03-28-2014
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Large-area arrays of vertically aligned ZnO-nanotapers with tailored taper angle and height are electrodeposited on planar Zn-plate via continuously tuning the Zn(NH3)4(NO3)2 concentration in the electrolyte. Experimental measurements reveal that the field-emission performance of the ZnO-nanotaper arrays is enhanced with the sharpness and height of the ZnO-nanotapers. Theoretically, the ZnO-nanotaper is simplified to a "charge disc" model, based on which the characteristic macroscopic field enhancement factor (?C) is quantified. The theoretically calculated ?C values are in good agreement with the experimental ones measured from arrays of ZnO-nanotapers with a series of geometrical parameters. The ZnO-nanotaper arrays have promising potentials in field-emission. The electrochemical synthetic strategy we developed may be extended to nanotaper arrays of other materials that are amenable to electrodeposition, and the "charge disc" model can be used for quasi-one-dimensional field emitters of other materials with nano-sized diameters.
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[IL-18 single nucleotide polymorphisms in hematologic malignances with HLA matched sibling donor allogeneic hematopoietic stem cell transplantation].
Zhonghua Xue Ye Xue Za Zhi
PUBLISHED: 03-27-2014
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To explore the impact of interleukin-18 (IL-18) single nucleotide polymorphisms on outcomes of hematologic malignances with HLA-matched sibling donor hematopoietic stem cell transplantation (allo-HSCT).
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Intermittent low-dose digoxin may be effective and safe in patients with chronic heart failure undergoing maintenance hemodialysis.
Exp Ther Med
PUBLISHED: 03-25-2014
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A low dose of digoxin is known to reduce mortality and hospitalization in patients with heart failure; however, the safety of digoxin in treating patients with heart failure on maintenance hemodialysis remains controversial. The objective of this study was to determine the effectiveness and safety of digoxin at lower doses in patients with heart failure on maintenance hemodialysis using a retrospective cohort study. This study included 67 heart-failure patients on maintenance hemodialysis: Twenty-four patients received intermittent low doses of digoxin (ILDD), 23 patients received continuous low doses of digoxin (CLDD) and the remaining patients were used as a control group without digoxin treatment. The brain natriuretic peptide (BNP) level and serum digoxin concentrations (SDCs) were measured by ELISA and the changes in left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF), cardiac output (CO) and heart rate (HR) were evaluated by two-dimensional echocardiography. The symptoms of digoxin toxicity were monitored in the treated patients. Compared with the control group, LVEDD, BNP and HR decreased significantly between days 0 and 60 in the ILDD and CLDD groups, but LVEF and CO increased between days 0 and 60 in the same groups (all P<0.05). The levels of BNP and the LVEDD, CO, LVEF and HR were not significantly different between the ILDD and CLDD groups (P>0.05). Furthermore, and the mean SDC of the ILDD group was lower than that of the CLDD group. In the ILDD group, no patients had apparent symptoms of toxicity, but four patients developed digoxin toxicity in the CLDD group. In conclusion an intermittent lower dose of digoxin has beneficial effects and clinical safety in hemodialysis patients with congestive heart failure.
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Pulsed electromagnetic fields partially preserve bone mass, microarchitecture, and strength by promoting bone formation in hindlimb-suspended rats.
J. Bone Miner. Res.
PUBLISHED: 03-09-2014
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A large body of evidence indicates that pulsed electromagnetic fields (PEMF), as a safe and noninvasive method, could promote in vivo and in vitro osteogenesis. Thus far, the effects and underlying mechanisms of PEMF on disuse osteopenia and/or osteoporosis remain poorly understood. Herein, the efficiency of PEMF on osteoporotic bone microarchitecture, bone strength, and bone metabolism, together with its associated signaling pathway mechanism, was systematically investigated in hindlimb-unloaded (HU) rats. Thirty young mature (3-month-old), male Sprague-Dawley rats were equally assigned to control, HU, and HU + PEMF groups. The HU + PEMF group was subjected to daily 2-hour PEMF exposure at 15 Hz, 2.4 mT. After 4 weeks, micro-computed tomography (µCT) results showed that PEMF ameliorated the deterioration of trabecular and cortical bone microarchitecture. Three-point bending test showed that PEMF mitigated HU-induced reduction in femoral mechanical properties, including maximum load, stiffness, and elastic modulus. Moreover, PEMF increased serum bone formation markers, including osteocalcin (OC) and N-terminal propeptide of type 1 procollagen (P1NP); nevertheless, PEMF exerted minor inhibitory effects on bone resorption markers, including C-terminal crosslinked telopeptides of type I collagen (CTX-I) and tartrate-resistant acid phosphatase 5b (TRAcP5b). Bone histomorphometric analysis demonstrated that PEMF increased mineral apposition rate, bone formation rate, and osteoblast numbers in cancellous bone, but PEMF caused no obvious changes on osteoclast numbers. Real-time PCR showed that PEMF promoted tibial gene expressions of Wnt1, LRP5, ?-catenin, OPG, and OC, but did not alter RANKL, RANK, or Sost mRNA levels. Moreover, the inhibitory effects of PEMF on disuse-induced osteopenia were further confirmed in 8-month-old mature adult HU rats. Together, these results demonstrate that PEMF alleviated disuse-induced bone loss by promoting skeletal anabolic activities, and imply that PEMF might become a potential biophysical treatment modality for disuse osteoporosis.
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[Outcome analysis of alternative donor allogeneic hematopoietic cell transplantation in the treatment of 19 severe aplastic anemia patients].
Zhonghua Xue Ye Xue Za Zhi
PUBLISHED: 03-08-2014
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To evaluate the efficacy of alternative donor allogeneic hematopoietic stem cell transplantation (AD allo-HSCT) in the treatment of severe aplastic anemia (SAA).
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Functional role of asparaginyl endopeptidase ubiquitination by TRAF6 in tumor invasion and metastasis.
J. Natl. Cancer Inst.
PUBLISHED: 03-07-2014
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Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive.
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Reduced miR-3127-5p expression promotes NSCLC proliferation/invasion and contributes to dasatinib sensitivity via the c-Abl/Ras/ERK pathway.
Sci Rep
PUBLISHED: 03-03-2014
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miR-3127-5p is a primate-specific miRNA which is down-regulated in recurrent NSCLC tissue vs. matched primary tumor tissue (N = 15) and in tumor tissue vs. normal lung tissue (N = 177). Reduced miR-3127-5p expression is associated with a higher Ki-67 proliferation index and unfavorable prognosis in NSCLC. Overexpression of miR-3127-5p significantly reduced NSCLC cells proliferation, migration, and motility in vitro and in vivo. The oncogene ABL1 was a direct miR-3127-5p target, and miR-3127-5p regulated the activation of the Abl/Ras/ERK pathway and transactivated downstream proliferation/metastasis-associated molecules. Overexpression of miR-3127-5p in A549 or H292 cells resulted in enhanced resistance to dasatinib, an Abl/src tyrosine kinase inhibitor. miR-3127-5p expression levels were correlated with dasatinib sensitivity in NSCLC cell lines without K-Ras G12 mutation. In conclusion, miR-3127-5p acts as a tumor suppressor gene and is a potential biomarker for dasatinib sensitivity in the non-mutated Ras subset of NSCLC.
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Capillary-based three-dimensional immunosensor assembly for high-performance detection of carcinoembryonic antigen using laser-induced fluorescence spectrometry.
Anal. Chem.
PUBLISHED: 01-22-2014
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A novel capillary-based three-dimensional (3D) fluoroimmunosensor for carcinoembryonic antigen detection was explored for the first time. The immunosensor was designed in symmetrical cylinder structure and fabricated with capillary tubes encapsulated in a quartz tube. The 3D design of the sensor increased the area of sensing surface, flexibility in light path design and efficiency of fluorescence collection by aluminum foil, resulting in analytical performance improvement. The CEA immunosensor was constructed in double antibody sandwich format. Fluorescence signals from DyLight 550-labeled antibody were measured using a laser-induced fluorescence spectrometry. There is an obvious improvement in the linear detection range of 0.7-80 ng/mL. This novel 3D immunosensor dramatically improved the detection limit (1.1 pmol/L CEA) and sensitivity. Assay validation studies indicated that the correlation coefficient reached 0.9935, with recoveries of 92.82-118.81%. Furthermore, the immunosensor was successfully applied to CEA determination in actual saliva specimens with high sensitivity and acceptable precision. Regarding accuracy, the results obtained by 3D immunosensor were not significantly different (t test) from those obtained by validated electrochemiluminescence immunoassay. This new 3D CEA immunosensor was demonstrated to be a high-performance tool for CEA diagnostics.
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Overexpression of microRNA-99a attenuates heart remodelling and improves cardiac performance after myocardial infarction.
J. Cell. Mol. Med.
PUBLISHED: 01-08-2014
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MicroRNAs are involved in the regulation of various cellular processes, including cell apoptosis and autophagy. Expression of microRNA-99a (miR-99a) is reduced in apoptotic neonatal mice ventricular myocytes (NMVMs) subjected to hypoxia. We hypothesize that miR-99a might restore cardiac function after myocardial infarction (MI) by up-regulation of myocyte autophagy and apoptosis. We observed down-regulated miR-99a expression in NMVMs exposed to hypoxia using TaqMan quantitative reverse transcriptase-polymerase chain reaction analysis (RT-PCR). We also observed that miR-99a overexpression decreased hypoxia-mediated apoptosis in cultured NMVMs. To investigate whether overexpression of miR-99a in vivo could improve cardiac function in ischaemic heart, adult C57/BL6 mice undergoing MI were randomized into two groups and were intra-myocardially injected with lenti-99a-green fluorescent protein (GFP) or lenti-GFP (control). Four weeks after MI, lenti-99a-GFP group showed significant improvement in both left ventricular (LV) function and survival ratio, as compared to the lenti-GFP group. Histological analysis, western blotting analysis and electron microscopy revealed decreased cellular apoptosis and increased autophagy in cardiomyocytes of lenti-99a-GFP group. Furthermore, western blotting analysis showed inhibited mammalian target of rapamycin (mTOR) expression in the border zones of hearts in miR-99a-treated group. Our results demonstrate that miR-99a overexpression improves both cardiac function and survival ratio in a murine model of MI by preventing cell apoptosis and increasing autophagy via an mTOR/P70/S6K signalling pathway. These findings suggest that miR-99a plays a cardioprotective role in post-infarction LV remodelling and increased expression of miR-99a may have a therapeutic potential in ischaemic heart disease.
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Retrotransposon "Qian" mediated segmental duplication in silkworm, Bombyx mori.
Insect Biochem. Mol. Biol.
PUBLISHED: 01-06-2014
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Transposable elements constitute a large fraction of the eukaryotic genomes. They have the potential to alter genome structure and play a major role in genome evolution. Here, we report a segmental duplication mediated by a novel long terminal repeat (LTR) retrotransposon as the cause of an egg-shell recessive lethal mutant (l-em mutant) in silkworm (Bombyx mori). The segmental duplication resulted in the duplication of six genes and the disruption of two genes. Disruption of BmEP80 (B. mori egg protein 80), a gene encoding a major egg-shell structure protein, is likely responsible for the lethal water-loss phenotype in the l-em/l-em mutant. Our data revealed that BmEP80 is present in the inner egg-shell layer and plays important roles in resistance to water efflux form eggs. A novel LTR retrotransposon (named as "Qian") was identified and the model for the Qian-mediated chromosomal segmental duplication was proposed. Detail biochemical and genomic analyses on the l-em mutant offer an opportunity to demonstrate that an LTR retrotransposon could trigger duplication of a chromosomal segment (?96.3 kb) and confer novel phenotype.
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Multifunctional nanoparticles via host-guest interactions: a universal platform for targeted imaging and light-regulated gene delivery.
Chem. Commun. (Camb.)
PUBLISHED: 01-03-2014
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Host-guest assembly provides a universal platform to construct responsive carrier systems for targeted imaging and controllable gene delivery. The best advantage of this strategy is that systems are very easy to handle, do not involve tedious chemical reactions and can be flexibly optimized by changing the functional tags responding to a request.
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Hemolytic Streptococcus may exacerbate kidney damage in IgA nephropathy through CCL20 response to the effect of Th17 cells.
PLoS ONE
PUBLISHED: 01-01-2014
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The exacerbation of IgA nephropathy (IgAN) is related to respiratory tract infection with hemolytic streptococcus (HS), but the mechanism is unknown. In this study we investigated the role of chemokine ligand 20 (CCL20) in response to the effect of T helper 17 (Th17) cells in the pathogenesis of IgAN associated with HS.
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Moderate-intensity rotating magnetic fields do not affect bone quality and bone remodeling in hindlimb suspended rats.
PLoS ONE
PUBLISHED: 01-01-2014
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Abundant evidence has substantiated the positive effects of pulsed electromagnetic fields (PEMF) and static magnetic fields (SMF) on inhibiting osteopenia and promoting fracture healing. However, the osteogenic potential of rotating magnetic fields (RMF), another common electromagnetic application modality, remains poorly characterized thus far, although numerous commercial RMF treatment devices have been available on the market. Herein the impacts of RMF on osteoporotic bone microarchitecture, bone strength and bone metabolism were systematically investigated in hindlimb-unloaded (HU) rats. Thirty two 3-month-old male Sprague-Dawley rats were randomly assigned to the Control (n?=?10), HU (n?=?10) and HU with RMF exposure (HU+RMF, n?=?12) groups. Rats in the HU+RMF group were subjected to daily 2-hour exposure to moderate-intensity RMF (ranging from 0.60 T to 0.38 T) at 7 Hz for 4 weeks. HU caused significant decreases in body mass and soleus muscle mass of rats, which were not obviously altered by RMF. Three-point bending test showed that the mechanical properties of femurs in HU rats, including maximum load, stiffness, energy absorption and elastic modulus were not markedly affected by RMF. µCT analysis demonstrated that 4-week RMF did not significantly prevent HU-induced deterioration of femoral trabecular and cortical bone microarchitecture. Serum biochemical analysis showed that RMF did not significantly change HU-induced decrease in serum bone formation markers and increase in bone resorption markers. Bone histomorphometric analysis further confirmed that RMF showed no impacts on bone remodeling in HU rats, as evidenced by unchanged mineral apposition rate, bone formation rate, osteoblast numbers and osteoclast numbers in cancellous bone. Together, our findings reveal that RMF do not significantly affect bone microstructure, bone mechanical strength and bone remodeling in HU-induced disuse osteoporotic rats. Our study indicates potentially obvious waveform-dependent effects of electromagnetic fields-stimulated osteogenesis, suggesting that RMF, at least in the present form, might not be an optimal modality for inhibiting disuse osteopenia/osteoporosis.
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The incidence and risk factors of meningitis after major craniotomy in China: a retrospective cohort study.
PLoS ONE
PUBLISHED: 01-01-2014
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Meningitis after neurosurgery can result in severe morbidity and high mortality. Incidence varies among regions and limited data are focused on meningitis after major craniotomy.
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Prevalence and predictors of mild cognitive impairment in Xi'an: a community-based study among the elders.
PLoS ONE
PUBLISHED: 01-01-2014
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Mild cognitive impairment (MCI) is an intermediate stage between normal cognitive function and dementia among aging individuals. This study was designed to estimate the prevalence of MCI and explore the possible risk factors including gender disparities among community-dwelling older individuals. The study was conducted in Xi'an, China. This is a cross-sectional study. A total of 815 individuals, 60 years and older were selected by stratified random cluster sampling. Cognitive function was measured using the mini-mental status examination (MMSE), the Chinese version of the Dementia Rating Scales (CDRS) was used to apply the diagnostic of non-dementia, and activities of daily living (ADL) and instrumental activities of daily living (IADL) systems were used to functional status. The association between sociodemographic characteristics, lifestyle, history of chronic diseases and MCI were evaluated separately for men and women using the Pearson ?²-test and binary logistic regression. Of the 815 community-dwelling individuals, 145 were found to have MCI. Overall, the prevalence of MCI was 18.5%, with a prevalence of 19.6% in women (105/535), and 15.3% (40/261) in men. The results of the binary logistical regression analysis indicated that age and history of stroke were associated with MCI in men. For women, the risk factors were lower level of educational and lack of religious attendance. Results suggested that the factors capable of influencing MCI differed profoundly between older men and older women. For this reason, different preventative measures should be adopted to delay or reverse cognitive impairment among community-dwelling older men and women.
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Epidermal Growth Factor Receptor Targeted Nuclear Delivery and High-Resolution Whole Cell X-ray Imaging of Fe3O4@TiO2 Nanoparticles in Cancer Cells.
ACS Nano
PUBLISHED: 11-27-2013
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Sequestration within the cytoplasm often limits the efficacy of therapeutic nanoparticles that have specific subcellular targets. To allow for both cellular and subcellular nanoparticle delivery, we have created epidermal growth factor receptor (EGFR)-targeted Fe3O4@TiO2 nanoparticles that use the native intracellular trafficking of EGFR to improve internalization and nuclear translocation in EGFR-expressing HeLa cells. While bound to EGFR, these nanoparticles do not interfere with the interaction between EGFR and karyopherin-?, a protein that is critical for the translocation of ligand-bound EGFR to the nucleus. Thus, a portion of the EGFR-targeted nanoparticles taken up by the cells also reaches cell nuclei. We were able to track nanoparticle accumulation in cells by flow cytometry and nanoparticle subcellular distribution by confocal fluorescent microscopy indirectly, using fluorescently labeled nanoparticles. More importantly, we imaged and quantified intracellular nanoparticles directly, by their elemental signatures, using X-ray fluorescence microscopy at the Bionanoprobe, the first instrument of its kind in the world. The Bionanoprobe can focus hard X-rays down to a 30 nm spot size to map the positions of chemical elements tomographically within whole frozen-hydrated cells. Finally, we show that photoactivation of targeted nanoparticles in cell nuclei, dependent on successful EGFR nuclear accumulation, induces significantly more double-stranded DNA breaks than photoactivation of nanoparticles that remain exclusively in the cytoplasm.
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Hadoop-GIS: A High Performance Spatial Data Warehousing System over MapReduce.
Proceedings VLDB Endowment
PUBLISHED: 11-05-2013
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Support of high performance queries on large volumes of spatial data becomes increasingly important in many application domains, including geospatial problems in numerous fields, location based services, and emerging scientific applications that are increasingly data- and compute-intensive. The emergence of massive scale spatial data is due to the proliferation of cost effective and ubiquitous positioning technologies, development of high resolution imaging technologies, and contribution from a large number of community users. There are two major challenges for managing and querying massive spatial data to support spatial queries: the explosion of spatial data, and the high computational complexity of spatial queries. In this paper, we present Hadoop-GIS - a scalable and high performance spatial data warehousing system for running large scale spatial queries on Hadoop. Hadoop-GIS supports multiple types of spatial queries on MapReduce through spatial partitioning, customizable spatial query engine RESQUE, implicit parallel spatial query execution on MapReduce, and effective methods for amending query results through handling boundary objects. Hadoop-GIS utilizes global partition indexing and customizable on demand local spatial indexing to achieve efficient query processing. Hadoop-GIS is integrated into Hive to support declarative spatial queries with an integrated architecture. Our experiments have demonstrated the high efficiency of Hadoop-GIS on query response and high scalability to run on commodity clusters. Our comparative experiments have showed that performance of Hadoop-GIS is on par with parallel SDBMS and outperforms SDBMS for compute-intensive queries. Hadoop-GIS is available as a set of library for processing spatial queries, and as an integrated software package in Hive.
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C70-carboxyfullerenes as efficient antioxidants to protect cells against oxidative-induced stress.
ACS Appl Mater Interfaces
PUBLISHED: 10-22-2013
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Oxidative stress induced by excessive production of reactive oxygen species (ROS) has been implicated in the etiology of many human diseases. Acquiring a highly efficient antioxidant with good biocompatibility is of significance in eliminating the deleterious effect induced by the oxidative stress. Herein, we address our efforts on investigating the cytoprotective effect of carboxyfullerenes on H2O2-injured cells. Meanwhile, the uptake and intracellular location of carboxyfullerenes were studied. The results show that C70-carboxyfullerenes (dimalonic acid C70 fullerene (DF70) and trimalonic acid C70 fullerene (TF70)) exhibit an obviously protective effect against oxidative stress on C2C12 cells at concentrations as low as 2.5 ?mol L(-1), whereas C60-carboxyfullerenes (dimalonic acid C60 fullerene (DF60) and quadri-malonic acid C60 fullerene (QF60)) show a protective effect at relatively higher concentration (40 ?mol L(-1)). The molecular structure of carboxyfullerenes and the physiological state of cells play an important role in the different cytoprotective capability. Further study reveals that DF70 and TF70 could enter into cells and mainly localize into the lysosome, which possibly involves the protective mechanism by stabilizing lysosome. The use of a significantly low concentration of C70-carboxyfullerene as the antioxidative agent will benefit the therapeutic approaches aiming at alleviating ROS-induced injuries such as muscle disorder and arthritis.
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Molecular cloning and characterization of the ?-chain gene of goose immunoglobulin heavy chain.
Biotechnol. Lett.
PUBLISHED: 10-15-2013
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A novel gene encoding the ?-chain of goose immunoglobulin heavy-chain (Ig?) was cloned by reverse transcription-PCR. The cDNA had 1,760 bp and encompassed a partial V-D-J region of the heavy chain, a constant region (C?) and 3-untranslated region of ?-chain. The C? gene contains four constant region domains (CH1-CH4). Phylogenetic analysis indicated that goose IgC? has a close genetic relationship with duck, ostrich and chicken IgC?. Three-dimensional modeling and glycosylation analysis revealed the goose Ig? is consistent with the characterization of immunoglobulin. Western blotting suggested the goose IgC? has the same antigenicity to natural IgA. In general, the identification of goose immunoglobulin not only provides insights into the evolution of the Ig heavy-chain gene family, but may also benefit future studies of the avian immune system.
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[Effect of calcitriol on secondary hyperparathyroidism].
Zhong Nan Da Xue Xue Bao Yi Xue Ban
PUBLISHED: 09-28-2013
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To investigate the calcium-phosphate metabolic condition in maintenance hemodialysis patients with chronic kidney disease (CKD), and to observe the effect of large dose calcitriol on secondary hyperparathyroidism (SHPT).
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Preparation and characterization of bionic bone structure chitosan/hydroxyapatite scaffold for bone tissue engineering.
J Biomater Sci Polym Ed
PUBLISHED: 09-23-2013
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Three-dimensional oriented chitosan (CS)/hydroxyapatite (HA) scaffolds were prepared via in situ precipitation method in this research. Scanning electron microscopy (SEM) images indicated that the scaffolds with acicular nano-HA had the spoke-like, multilayer and porous structure. The SEM of osteoblasts which were polygonal or spindle-shaped on the composite scaffolds after seven-day cell culture showed that the cells grew, adhered, and spread well. The results of X-ray powder diffractometer and Fourier transform infrared spectrometer showed that the mineral particles deposited in the scaffold had phase structure similar to natural bone and confirmed that particles were exactly HA. In vitro biocompatibility evaluation indicated the composite scaffolds showed a higher degree of proliferation of MC3T3-E1 cell compared with the pure CS scaffolds and the CS/HA10 scaffold was the highest one. The CS/HA scaffold also had a higher ratio of adhesion and alkaline phosphate activity value of osteoblasts compared with the pure CS scaffold, and the ratio increased with the increase of HA content. The ALP activity value of composite scaffolds was at least six times of the pure CS scaffolds. The results suggested that the composite scaffolds possessed good biocompatibility. The compressive strength of CS/HA15 increased by 33.07% compared with the pure CS scaffold. This novel porous scaffold with three-dimensional oriented structure might have a potential application in bone tissue engineering.
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Solution Structure of Energy Stored System I: Aqua-B(OH)4(-): A DFT, Car-Parrinello Molecular Dynamics, and Raman Study.
J Phys Chem B
PUBLISHED: 09-23-2013
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A systematic study on the structure, stability, and Raman spectra of the metaborate anion hydrated clusters, B(OH)4(-)(H2O)n, (n = 1-15) was carried out by DFT in both gaseous and aqueous phase at the B3LYP/aug-cc-pVDZ level; all of these stable configurations were described, and the most stable hydrated clusters were chosen. The hydrogen bonds in those hydrated clusters were described in three different items: symmetrical double hydrogen bonding (DHB), single hydrogen bonding (SHB), and interwater hydrogen bonding (WHB). The distance of SHB is shorter than that of DHB, and multiple SHBs are more stable than a single DHB. In small size clusters (n ? 5), a structure with more DHBs is more stable than other arrangements. With continued increase in size, more SHBs were found in the first hydration sphere: when n ? 9, only SHBs can be found, and when n ? 12, a full hydration structure is formed with 12 SHBs and a hydration number of 10-12. The Car-Parrinello molecular dynamics simulation shows that only the first hydration sphere can be found, and the hydration number of B(OH)4(-) is 9.2 and the hydration distance is 3.68. The total symmetrical stretching vibration of B(OH)4(-) in hydrated B(OH)4(-)(H2O)n is blue shifted with increasing cluster size. After consideration of hydration, the calculated characteristic frequencies are in accord with the experiment characteristic frequency of B(OH)4(-).
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Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increase ?ENaC transcription.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 09-11-2013
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Aldosterone is a major regulator of Na(+) absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na(+) channel (ENaC). MR(-/-) mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates ?ENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the ?ENaC promoter and upregulation of ?ENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR(-/-) mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR(-/-) vs. MR(+/+) mice had significantly lower ?ENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the ?ENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR(-/-) mice.
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Fluorescent labels in biosensors for pathogen detection.
Crit. Rev. Biotechnol.
PUBLISHED: 07-27-2013
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Abstract Infectious diseases caused by pathogens have become a life-threatening problem for millions of people around the world in recent years. Therefore, the need of efficient, fast, low-cost and user-friendly biosensing systems to monitor pathogen has increased enormously in the last few years. This paper presents an overview of different fluorescent labels and the utilization of fluorescence-based biosensor techniques for rapid, direct, sensitive and real-time identification of bacteria. In these biosensors, organic dyes, nanomaterials and rare-earth elements are playing an increasing role in the design of biosensing systems with an interest for applications in bacterial analysis.
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Distinct roles of sAPP-? and sAPP-? in regulating U251 cell differentiation.
Curr Alzheimer Res
PUBLISHED: 07-21-2013
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Sequential cleavages of APP by ?-secretase and ?-secretase release ?-amyloid (A?) and one secreted form of APP (sAPP-?) in Alzheimer s disease (AD). Alternatively, in non-pathological situations, APP is predominantly cleaved by ?-secretase within the amyloid sequence, to release the other soluble form of APP, sAPP-?. However, the functions of the two types of sAPP are still unclear. We performed this study to compare the function of sAPP-? and sAPP-? in differentiation of the glioma cell line U251. We found that sAPP-? suppressed astrocytic differentiation and promoted neuronal differentiation in U251 cells. Additionally, sAPP-? enhanced U251 terminal differentiation into a cholinergic-like neuronal phenotype. In contrast, sAPP-? suppressed neuronal differentiation and promoted the astrocytic differentiation of U251 cells. These findings could not only enrich the knowledge of the potential physiological function of sAPP-? and sAPP-?, but also indicate that they may be connected to the pathological mechanism of AD. Furthermore, these findings suggest that new strategies, such as increasing the level of sAPP-? and/or decreasing the level of sAPP-? in brain, or transplanting stem cells with increased sAPP-? and/or decreased sAPP-?, may have potential value for AD treatment.
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Inhibition of Rho-kinase alleviates peritoneal fibrosis and angiogenesis in a rat model of peritoneal dialysis.
Ren Fail
PUBLISHED: 07-18-2013
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The present study investigated whether Rho-kinase inhibition had a therapeutic role on the pathogenesis of peritoneal fibrosis and angiogenesis.
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Renoprotective effect of ramulus mori polysaccharides on renal injury in STZ-diabetic mice.
Int. J. Biol. Macromol.
PUBLISHED: 07-17-2013
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Recently, increasing evidences have suggested that inflammatory stress is markedly occurred in the impaired tissue. Thus, the streptozotocin (STZ)-induced diabetic mice were used to investigate the potential renoprotective effect of ramulus mori polysaccharides (RMP) and to discuss the underlying mechanism. The results from the present study showed that RMP significantly lowered the blood glucose and serum levels of glycosylated protein, cholesterol, urea nitrogen (Urea-N), creatinine (Cr) and 24-hour urine protein, while the albumin content was elevated. Meanwhile, the proinflammatory cytokines such as interleukin-6 (IL-6), interferon-gamma (IFN-?) and tumor necrosis factor alpha (TNF-?) in renal tissue of STZ-lesioned mice were reduced by RMP treatment. Similarly, pathological examination indicated that STZ-induced renal injury was effectively mitigated. In addition, the protein levels of interleukin-1 (IL-1) and interleukin-1 receptor (IL-1R) in renal tissue were notably decreased. Moreover, the endogenous expressions of phosphorylated-I?B (p-I?B) and nuclear factor-kappa B (NF-?B) were down-regulated, respectively. Together, these findings revealed that RMP treatment effectively attenuated STZ-induced cytotoxicity in renal tissue, in which RMP-exerted renoprotection was associated with intrarenally debilitating inflammation reaction through blocking the IL-1/NF-?B pathway, thereby maintaining the renal homeostasis.
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Smad7 gene transfer attenuates angiogenesis in peritoneal dialysis rats.
Nephrology (Carlton)
PUBLISHED: 07-03-2013
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Transforming growth factor-? (TGF-?) has been shown to play a role in peritoneal angiogenesis associated with peritoneal dialysis (PD). The present study investigated whether blockade of TGF-? signalling with Smad7 has a therapeutic effect on PD induced-peritoneal angiogenesis.
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Fullerene-induced increase of glycosyl residue on living plant cell wall.
Environ. Sci. Technol.
PUBLISHED: 06-11-2013
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In this work, we have investigated the change of cell wall for the tobacco plant cell (Nicotiana tobacum L. cv. Bright Yellow) under the repression of water-soluble carboxyfullerenes (C70(C(COOH)2)(2-4)). The adsorption of C70(C(COOH)2)(2-4) on cell wall led to the disruption of cell wall and membrane, and consequently, cell growth inhibition. Results from atomic force microscopy (AFM) force measurement and confocal imaging revealed an increase of the glycosyl residue on the cell wall of carboxyfullerene-treated cells, with a time- and dose-dependent manner, and accompanied by the elevated reactive oxygen species (ROS). Moreover, the stimulation-sensitive alteration of glycosyl residue and ROS was demonstrated, which suggested a possible protection strategy for the plant cells under fullerene repression. This study provides the first direct evidence on the change of plant cell wall composition under the repression of fullerene and is the first successful application of AFM ligand-receptor binding force measurement to the living plant cell. The new information present here would help to a better understanding and assessment of the biological effect of fullerenes on plant.
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Nanocontainers made of various materials with tunable shape and size.
Sci Rep
PUBLISHED: 06-07-2013
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Nanocontainers have great potentials in targeted drug delivery and nanospace-confined reactions. However, the previous synthetic approaches exhibited limited control over the morphology, size and materials of the nanocontainers, which are crucial in practical applications. Here, we present a synthetic approach to multi-segment linear-shaped nanopores with pre-designed morphologies inside anodic aluminium oxide (AAO), by tailoring the anodizing duration after a rational increase of the applied anodizing voltage and the number of voltage increase during Al foil anodization. Then, we achieve nanocontainers with designed morphologies, such as nanofunnels, nanobottles, nano-separating-funnels and nanodroppers, with tunable sizes and diverse materials of carbon, silicon, germanium, hafnium oxide, silica and nickel/carbon magnetic composite, by depositing a thin layer of materials on the inner walls of the pre-designed AAO nanopores. The strategy has far-reaching implications in the designing and large-scale fabrication of nanocontainers, opening up new opportunities in nanotechnology applications.
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Temporal variations of benthic diatom community and its main influencing factors in a subtropical river, China.
Environ Sci Pollut Res Int
PUBLISHED: 05-31-2013
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Benthic diatoms are the main component in many aquatic ecosystems such as streams, creeks and rivers, and they function as important primary producers and chemical modulators for other organisms in the ecosystems. In this study, the composition of benthic diatoms was investigated and further explored the primary physicals and chemicals affecting their temporal variations in the upper Han River, China. There were seasonal variations in physical and chemical variables in waters over the sampling period of 2007-2010. Water temperature (t), chemical oxygen demand, total nitrogen, dissolved organic carbon (DOC), silica and fluoride were much higher in the high flow season (i.e., July or August) than these in the base flow season. Three species Achnanthidium minutissimum (composed of 10.7 % of the total diatom abundance), Achnanthidium pyrenaicum (11.9 %), and Achnanthidium subatomus (12.7 %) accounting for more than 5 % of the total diatom abundance were persistently dominant in all seasons, while the other two prostrate and motile species including Eolimna minima and Nitzschia dissipata also dominant in the base flow season. The species richness always peaked in autumn with significant difference with summer (p?
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[Effect of Cordyceps sinensis on expressions of HIF-1? and VEGF in the kidney of rats with diabetic nephropathy].
Zhong Nan Da Xue Xue Bao Yi Xue Ban
PUBLISHED: 05-31-2013
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To examine the expressions of hypoxia inducible factor-1? (HIF-1?) and vascular endothelial growth factor (VEGF) in the kidney of rats with diabetic nephropathy before and after the treatment of Cordyceps sinensis, and to explore the mechanism of Cordyceps sinensis against hypoxia.
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Diversity of lactic acid bacteria during fermentation of a traditional Chinese fish product, Chouguiyu (stinky mandarinfish).
J. Food Sci.
PUBLISHED: 05-27-2013
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Chouguiyu, or stinky mandarinfish, is a traditional Chinese fermented fish product made of mandarinfish by spontaneous fermentation at the anaerobic condition with low-salt concentration. In order to get a primary understanding of the microbial community presenting in the Chouguiyu fermentation, 61 cultures of lactic acid bacteria (LAB) from various fermentation period were isolated using MRS agar plates and characterized based on a combination of phenotypic and genotypic approaches including amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA partial gene sequencing analysis. Eight distinct bacterial species belonging to 6 genera were identified in total. Among them, Lactobacillus sakei was the dominant species (63%) during the fermentation, which exhibited great variety in phenotypic tests but unique genotypic characters. Meanwhile, the other LAB species including Lactococcus (Lc.) garvieae, Lc. lactis, Lc. raffinolactis, Vagococcus sp., Enterococcus hermanniensis, Macrococcus caseolyticus as well as Streptococcus parauberis were also recovered from the different fermentation periods, especially at the initial point of the fermentation. This seems to be the 1st report investigating the LAB composition involved in Chouguiyu fermentation and the data obtained in this study may be valuable for selecting starter culture for Chouguiyu industrial-scale production.
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Long-Term Fluorescent Cellular Tracing by the Aggregates of AIE Bioconjugates.
J. Am. Chem. Soc.
PUBLISHED: 05-22-2013
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There is a great demand for long-term cellular tracers because of their great importance in monitoring biological processes, pathological pathways, therapeutic effects, etc. Herein we report a new type of fluorescence "turn-on" probe for tracing live cells over a long period of time. We synthesized the fluorogenic probe by attaching a large number of tetraphenylethene (TPE) labels to a chitosan (CS) chain. The resultant TPE-CS bioconjugate shows a unique aggregation-induced emission (AIE) behavior. It is nonfluorescent when dissolved but becomes highly emissive when its molecules are aggregated. The AIE aggregates can be readily internalized by HeLa cells. The cellular staining by the TPE-CS aggregates is so indelible that it enables cell tracing for as long as 15 passages. The internalized AIE aggregates are kept inside the cellular compartments and do not contaminate other cell lines in the coculture systems, permitting the differentiation of specific cancerous cells from normal healthy cells.
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Evaluation of nutrient removal efficiency and microbial enzyme activity in a baffled subsurface-flow constructed wetland system.
Bioresour. Technol.
PUBLISHED: 05-21-2013
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In this study, the enzyme activities and their relationships to domestic wastewater purification are investigated in four different types of subsurface-flow constructed wetlands (CWs), namely the traditional horizontal subsurface-flow, horizontal baffled subsurface-flow, vertical baffled subsurface-flow, and composite baffled subsurface-flow CWs. Results showed that the urease activity in the composite baffled subsurface-flow CW was significantly higher than in the other three CWs, while the phosphatase activity in the vertical baffled subsurface-flow CW were higher than in the other three CWs. There were significant and very significant correlations between the activities of urease and the removal rates of TN and NH4(+)-N for the horizontal baffled flow, horizontal subsurface flow, and composite baffled subsurface flow CWs. This study suggests that the activity of urease in the root zones of those three CWs is an important indicator for N purification from wastewaters.
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Study on the regulation of cell adhesion molecule expression and function in placenta from women with intrahepatic cholestasis of pregnancy.
Med. Hypotheses
PUBLISHED: 05-19-2013
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Intrahepatic cholestasis of pregnancy (ICP) is a common complication of pregnancy manifested as skin pruritus of cholestasis. ICP occurs mainly in the second or third trimester of pregnancy and may cause fetal distress, unexpected intrauterine fetal death and does serious harm to maternal and fetal health. The pathogenesis of ICP is still unclear. In ICP placentas, placental syncytiotrophoblasts are the most direct contact between maternal high bile acid environment and fetus. Our previous study found that in ICP placental syncytiotrophoblasts, both mRNA expression level and protein expression level of vascular cell adhesion molecule-1 (VCAM-1), were significantly elevated. Since VCAM-1 is important in inflammatory injury of lymphocytes, we speculate that ICP pathogenesis may be associated with VCAM-1 up-regulation which may lead to inflammatory injury and cause intrauterine fetal distress, intrauterine fetal death and other adverse outcomes. Elucidation of this mechanism should help reveal the ICP pathogenesis and facilitate the clinical treatment of intrauterine fetal death.
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Effects of legumain as a potential prognostic factor on gastric cancers.
Med. Oncol.
PUBLISHED: 05-11-2013
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Although legumain has been found to be a prognostic factor in both breast cancer and colorectal cancer, its effects on gastric cancer are unknown. In this study, we investigated effects of legumain on gastric cancer and the correlation between legumain expression and prognosis of gastric cancer patients. SGC7901 cells were transduced with legumain cDNA (SGC7901-hLeg) for overexpression of legumain or with legumain shRNA to knock down legumain. In vitro tumor migration was examined by wound healing assay. Furthermore, a tumorigenicity and metastasis mouse model was used to examine legumain function in vivo; asparaginyl endopeptidase inhibitor (AEPI, an inhibitor of legumain) was injected to the mice (i.p.) to evaluate its therapeutic effect. Tissue microarray analysis from 112 gastric cancer patients was performed to evaluate the association between legumain expression and the cumulative survival time. Legumain was highly expressed in gastric cancer patients and some gastric cancer cell lines. Legumain promoted gastric cell migration in vitro and promoted gastric tumor growth and metastasis in vivo, and these effects were reversed by knockdown of legumain with shRNA or treated with AEPI. In gastric cancer clinical samples, legumain expression in tumor was significantly higher than in non-tumor and was negatively associated with the cumulative survival rate. In conclusion, legumain was highly expressed in gastric adenocarcinoma; legumain promoted gastric cancer tumorigenesis and metastasis in vitro and in vivo. Legumain expression in tumor was a poor prognostic factor for gastric cancer patients, and legumain could be a potential target molecule for gastric cancer therapy in clinic.
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Puerarin, isolated from Pueraria lobata (Willd.), protects against hepatotoxicity via specific inhibition of the TGF-?1/Smad signaling pathway, thereby leading to anti-fibrotic effect.
Phytomedicine
PUBLISHED: 04-18-2013
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Recently, the TGF-?1/Smad signaling pathway has been investigated in the pathogenesis of hepatofibrosis, and pharmacological treatment of liver fibrosis targeted this pathway to determine its contribution to the inhibition of fibrotic development. Importantly, ethnopharmacology-derived Pueraria lobata has been reported to effectively reverse the fibrotic process in the liver. In the present study, we performed dimethylnitrosamine (DMN)-induced liver fibrosis in rats to assess the benefits of puerarin (PR), which was isolated from Pueraria lobata (Willd.), on ECM-derived hepatocytes associated with the TGF-?1/Smad pathway. Our results showed that the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN), type III precollagen (PCIII) and type IV collagen (CIV) were significantly reduced by PR treatment, while hepatic homogenates showed decreased levels of hydroxyproline (Hyp) and collagen I (Col I). Massons trichrome staining indicated that the DMN-induced liver fibrosis was alleviated. In addition, the protein expression levels of transforming growth factor-? l (TGF-? l), smad2, smad3, ?-SMA and TIMP-1 were downregulated specifically by PR treatment, whereas the protein expression levels of smad7 and MMP-1 were upregulated. Furthermore, we evaluated the PR-mediated inhibitory effect on TGF-?1-treated proliferation and activation in a rat liver stellate cell line (HSC-T6). These data resulted in inhibition of the cell growth of HSC-T6 in a dose-dependent manner and a reduction in T?RI, smad2 and smad3 expressed proteins in the presence of PR on TGF-?1-treated HSC-T6 cells, while smad7 levels were downregulated. Taken together, these findings identify a unique effect for PR-regulation of the TGF-?1/Smad pathway in blocking fibrotic development and provide a promising strategy for hepatofibrosis treatment.
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Plasma-enhanced antibody immobilization for the development of a capillary-based carcinoembryonic antigen immunosensor using laser-induced fluorescence spectroscopy.
Anal. Chem.
PUBLISHED: 04-18-2013
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In this study, antibody immobilization using a microwave-induced H2O/Ar plasma pretreatment was achieved for the first time. Plasma was used to activate the surface of a capillary-based immunosensor by increasing the density of silicon hydroxyls and dangling bonds to ensure better silanization. The capture antibodies were covalently immobilized after the silanized surface reacted with glutaraldehyde and antibodies. A Cy3-labeled detection antibody was used in combination with the antigen captured by the immunosensor to complete the sandwich-type immunoassay, and the signals were measured using a laser-induced fluorescence system. Microwave-induced H2O/Ar plasma pretreatment of the carcinoembryonic antigen (CEA) immunosensor improved the antibody immobilization, and there was an obvious improvement in the linear detection range, i.e., 1 order of magnitude compared with a commercial enzyme-linked immunosorbent assay (ELISA). This novel immobilization method dramatically improved the detection limit (0.5 pmol/L CEA) and sensitivity. Assay validation studies indicated that the correlation coefficient reached 0.9978, and the relative standard deviations were <7% for all samples, with recoveries of 99.7-107.1%. Furthermore, the immunosensor was applied successfully to CEA determination in actual saliva specimens with high sensitivity, acceptable precision, and reasonable accuracy. This enhanced CEA immunosensor based on microwave-induced H2O/Ar plasma was demonstrated to be a sensitive tool for CEA diagnostics.
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Atomic force microscopy study of the effects of water-soluble fullerenes on the elasticity of living plant cells.
Chem Asian J
PUBLISHED: 04-15-2013
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In this work, atomic force microscopy (AFM) was employed to characterize the elastic properties of a living suspension of Nicotiana tabacum L. cv. Bright Yellow (BY-2) cells and to investigate the changes in plant-cell elasticity that were induced by water-soluble C70 fullerene derivatives. The results revealed different effects of the three fullerene derivatives that had different numbers of carboxylic groups on the cell elasticity. BY-2 cells that were repressed by dimalonic-acid-modified C70 fullerenes (DiF70) and trimalonic-acid-modified C70 fullerenes (TriF70) showed a clear decrease in their Youngs modulus. However, the Youngs modulus of cells that were treated with tetramalonic-acid-modified C70 fullerenes (TetraF70) increased. Disruption of the actin cytoskeleton arrangement was observed following treatment with DiF70 and TriF70, but not with TetraF70. Moreover, the fullerene-induced cell-elasticity change was consistent with the change in cell-proliferation rate. This work provides a new approach and valuable information for the study of the biological effect of nanomaterials on plant cells.
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