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Find video protocols related to scientific articles indexed in Pubmed.
Post-synthesis DNA modifications using a trans-cyclooctene click handle.
Org. Biomol. Chem.
PUBLISHED: 11-20-2014
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Post-synthesis DNA modification is a very useful method for DNA functionalization. This is achieved by using a modified NTP, which has a handle for further modifications, replacing the corresponding natural NTP in polymerase-catalyzed DNA synthesis. Subsequently, the handle can be used for further functionalization after PCR, preferably through a very fast reaction. Herein we describe polymerase-mediated incorporation of trans-cyclooctene modified thymidine triphosphate (TCO-TTP). Subsequently, the trans-cyclooctene group was reacted with a tetrazine tethered to other functional groups through a very fast click reaction. The utility of this DNA functionalization method was demonstrated with the incorporation of a boronic acid group and a fluorophore. The same approach was also successfully used in modifying a known aptamer for fluorescent labelling applications.
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Synthesis, Structure, and SAR of Tetrahydropyran-Based LpxC Inhibitors.
ACS Med Chem Lett
PUBLISHED: 11-13-2014
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In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization.
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[Survival analysis of AIDS patients in Liangshan prefecture, Sichuan province from 1995 to 2012].
Zhonghua Yu Fang Yi Xue Za Zhi
PUBLISHED: 11-13-2014
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To analyze the survival time and its related factors among AIDS patients in Liangshan prefecture of Sichuan province from 1995 to 2012.
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Experimental demonstration of free-space based 120??Gb/s reconfigurable card-to-card optical interconnects.
Opt Lett
PUBLISHED: 11-01-2014
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In this Letter, we propose and experimentally demonstrate a free-space based reconfigurable card-to-card optical interconnect architecture with 16-carrierless-amplitude-phase modulation. Experimental results show that up to 120 Gb/s (3×40??Gb/s) flexible interconnection can be achieved for up to 30 cm distance with a worst-case receiver sensitivity of -9.70??dBm.
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A one-step route to Ag nanowires with a diameter below 40 nm and an aspect ratio above 1000.
Chem. Commun. (Camb.)
PUBLISHED: 10-18-2014
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The synthesis of long and thin Ag nanowires is important to achieve high performance transparent conductive films. We report a one-step route to synthesizing Ag nanowires with an average diameter of ?25 nm and an aspect ratio larger than 1000 by utilizing a mixture of poly(vinylpyrrolidone) with different molecular weights as the capping agent in a polyol reduction reaction.
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The complete mitochondrial genome of the endangered Hucho hucho (Salmonidae: Huchen).
Mitochondrial DNA
PUBLISHED: 10-17-2014
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Abstract Hucho hucho, one of the most endangered members of the family Salmonidae, is endemic to the Danube basin. In this study, the complete mitochondrial genome of H. hucho was sequenced and characterized. The genome is 16,751?bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a noncoding control region. The gene arrangement and nucleotide composition of the mitochondrial genome are similar to those of H. bleekeri. An 82?bp tandem repeat was identified in the control region, which is variable in length and copy number of repeat between and within species. The complete mitochondrial DNA of H. hucho should be useful to study population genetics, biogeography and adaptive evolution of this lineage.
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Application of Intravascular Ultrasound in the Emergency Diagnosis and Treatment of Patients with ST-Segment Elevation Myocardial Infarction.
Echocardiography
PUBLISHED: 10-08-2014
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This study aimed to examine the application of intravascular ultrasound (IVUS) in ST-segment elevation myocardial infarction (STEMI) patients with high thrombus burden (thrombus grade ?3) undergoing emergency diagnosis and primary percutaneous coronary intervention.
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Rational design of signal-on biosensors by using photoinduced electron transfer between Ag nanoclusters and split G-quadruplex halves-hemin complexes.
Chem. Commun. (Camb.)
PUBLISHED: 10-07-2014
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Photoinduced electron transfer (PET) between DNA-Ag nanoclusters (AgNCs) and G-quadruplex halves-hemin has been used for building a new sensing platform for the signal-on detection of adenosine and RNA.
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Measurements of multiphoton action cross sections for multiphoton microscopy.
Biomed Opt Express
PUBLISHED: 10-01-2014
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We report quantitative measurements of two-, three-, and four-photon excitation action cross sections of several commonly used fluorophores and fluorescent proteins at three different excitation wavelengths of 800 nm, 1300 nm, and 1680 nm. The measured cross section values are consistent with simple quantum mechanic estimations. These values indicate that the optimum repetition rate for deep tissue 3-photon microscopy is approximately 1 to 2 MHz. We further demonstrate that it is feasible to perform 4-photon fluorescence microscopy of GFP labeled microglia in mouse brain in vivo at 1700 nm. 4-photon excitation increases the accessibility of fluorophores at the long wavelength spectral window of 1700 nm.
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Uniform core-shell photonic crystal microbeads as microcarriers for optical encoding.
Langmuir
PUBLISHED: 10-01-2014
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We demonstrate a rapid and robust method to fabricate uniform core-shell photonic crystal (PC) microbeads by the microfluidic and centrifugation-redispersion technique. Colored crystalline colloidal arrays (CCAs) were first prepared through centrifugation-redispersion approach by self-assembly of polystyrene-poly(N-isopropylacrylamide) (PS-PNIPAm) core/shell nanoparticles (NPs). Different from the conventional NPs (e.g., charged PS or PNIPAm NPs), PS-PNIPAm NPs could easily self-assemble into well-ordered CCAs by only one purification step without laborious pretreatment (e.g., dialysis or ion exchange) or slow solvent-evaporation process. The CCAs is then encapsulated into a transparent polymer shell with functional groups (e.g., copolymer of ETPTA and butyl acrylate (BA)), triggering the formation of core-shell PC microbeads which can be used as optical encoding microcarriers. Importantly, this technique allows us to produce core-shell PC microbeads in a rapid and robust way, and the optical reflections of the PC microbeads appear highly stable to various external stimuli (e.g., temperature, pH value, and ionic strength) relying on the features of the CCAs core and protection of the polymer shell. Moreover, various probe biomolecules (e.g., proteins, antibodies, and so on) can be easily linked on the surface of the core-shell PC microbeads owing to the hydrophilic modification induced by the hydrolysis of BA on the microbead surface, enabling the multiplex biomolecular detection.
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Impact of copper overpressure on the synthesis of hexagonal boron nitride atomic layers.
ACS Appl Mater Interfaces
PUBLISHED: 09-22-2014
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Hexagonal boron nitride (h-BN) atomic layers are synthesized on polycrystalline copper foils via a novel chemical vapor deposition (CVD) process that maintains a vapor-phase copper overpressure during growth. Compared to h-BN films grown without a copper overpressure, this process results in a >10× reduction of 3-dimensional BN fullerene-like surface features, a reduction of carbon and oxygen contamination of 65% and 62%, respectively, an increase in h-BN grain size of >2×, and an 89% increase in electrical breakdown strength.
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Sensitive and selective amplified visual detection of cytokines based on exonuclease III-aided target recycling.
Chem. Commun. (Camb.)
PUBLISHED: 09-18-2014
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A new Exo III assisted strand-cleavage cycle and ligand-responsive quadruplex formation strategy for amplified and label-free detection of IFN-? was reported with a detection limit of 0.1 pM and a visual detection limit of 20 pM by the naked eye.
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Curcumin inhibits aerobic glycolysis and induces mitochondrial-mediated apoptosis through hexokinase II in human colorectal cancer cells in vitro.
Anticancer Drugs
PUBLISHED: 09-16-2014
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Curcumin, the major pigment of the dietary spice turmeric, has the potential for chemoprevention by promotion of apoptosis. Here, we investigated the molecular mechanisms of curcumin in glycolytic inhibition and apoptotic induction in human colorectal cancer HCT116 and HT29 cells. On the one hand, curcumin downregulated the expression and activity of hexokinase II (HKII) in HCT116 and HT29 cells in a concentration-dependent manner, but had little effect on the other key glycolytic enzymes (PFK, PGM, and LDH). On the other, curcumin induced dissociation of HKII from the mitochondria, resulting in mitochondrial-mediated apoptosis. Furthermore, the phosphorylation of mitochondrial HKII through AKT was responsible for the curcumin-induced dissociation of HKII, which was different from the mechanism of HKII inhibitor 3-BrPA. These results have important implications for the metabolism reprogramming effect and the susceptibility to curcumin-induced mitochondrial cytotoxicity through the regulation of HKII, and provide a molecular basis for the development of naturally compounds as novel anticancer agents for colorectal carcinoma.
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RASAL2 down-regulation in ovarian cancer promotes epithelial-mesenchymal transition and metastasis.
Oncotarget
PUBLISHED: 09-13-2014
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Ovarian cancer is the most lethal gynecologic malignancy, and transcoelomic metastasis is responsible for the greatest disease mortality. Although intensive efforts have been made, the mechanism behind this process remains unclear. RASAL2 is a GTPase activating proteins (GAPs) which was recently reported as a tumor suppressor in breast cancer. In this study, we identified RASAL2 as a regulator of epithelial-mesenchymal transition (EMT) and metastasis in ovarian cancer. RASAL2 was down-regulated in ovarian cancer samples compared with normal tissue samples, especially in advanced stages and grades. RASAL2 knockdown in ovarian cancer cell lines promoted in vitro anchorage-independent growth, cell migration and invasion and in vivo tumor formation. Moreover, we observed EMT in RASAL2-depleted cells. E-cadherin-mediated cell-cell adhesion was attenuated, and mesenchymal markers were up-regulated. Further investigation revealed that the oncogenic role of RASAL2 down-regulation was mediated by the Ras-ERK pathway. RASAL2 knockdown activated the Ras-ERK pathway, and inhibition of the pathway reversed the functional effects of RASAL2 depletion. Together, our results implicate RASAL2 as an EMT regulator and tumor suppressor in ovarian cancer, and down-regulation of RASAL2 promotes ovarian cancer progression.
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Graph Theory-Based Approach for Stability Analysis of Stochastic Coupled Systems With Lévy Noise on Networks.
IEEE Trans Neural Netw Learn Syst
PUBLISHED: 09-13-2014
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In this paper, a novel class of stochastic coupled systems with Lévy noise on networks (SCSLNNs) is presented. Both white noise and Lévy noise are considered in the networks. By exploiting graph theory and Lyapunov stability theory, criteria ensuring pth moment exponential stability and stability in probability of these SCSLNNs are established, respectively. These principles are closely related to the topology of the network and the perturbation intensity of white noise and Lévy noise. Moreover, to verify the theoretical results, stochastic coupled oscillators with Lévy noise on a network and stochastic Volterra predator-prey system with Lévy noise are performed. Finally, a numerical example about oscillators' network is provided to illustrate the feasibility of our analytical results.
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Optimization of the integrated citric acid-methane fermentation process by air stripping and glucoamylase addition.
Bioprocess Biosyst Eng
PUBLISHED: 09-07-2014
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To solve the problem of extraction wastewater in citric acid industry, an integrated citric acid-methane fermentation process was proposed. In the integrated process, extraction wastewater was treated by mesophilic anaerobic digestion and then reused to make mash for the next batch of citric acid fermentation. In this study, an Aspergillus niger mutant strain exhibiting resistance to high metal ions concentration was used to eliminate the inhibition of 200 mg/L Na(+) and 300 mg/L K(+) in anaerobic digestion effluent (ADE) and citric acid production increased by 25.0 %. Air stripping was used to remove ammonium, alkalinity, and part of metal ions in ADE before making mash. In consequence, citric acid production was significantly improved but still lower by 6.1 % than the control. Results indicated that metal ions in ADE synergistically inhibited the activity of glucoamylase, thus reducing citric acid production. When 130 U/g glucoamylase was added before fermentation, citric acid production was 141.5 g/L, which was even higher than the control (140.4 g/L). This process could completely eliminate extraction wastewater discharge and reduce water resource consumption.
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The relationship between the plant-encoded RNA-dependent RNA polymerase 1 and alternative oxidase in tomato basal defense against Tobacco mosaic virus.
Planta
PUBLISHED: 09-05-2014
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Salicylic acid (SA) plays a critical role in plant defense against pathogen attack. The SA-induced viral defense in plants is distinct from the pathways mediating bacterial and fungal defense, which is pathogenesis-related protein-independent but involves an RNA-dependent RNA polymerase 1 (RDR1)-mediated RNA silencing mechanism and/or an alternative oxidase (AOX)-associated defense pathway. However, the relationship between these two viral defense-related pathways remains unclear. In this study, Tobacco mosaic virus (TMV) inoculation onto Solanum lycopersicum (tomato) leaves induced a rapid induction of the SlAOX1a transcript level as well as the total and CN-resistant respiration at 0.5 dpi, followed by an increase in SlRDR1 gene expression at 1 dpi in the upper uninoculated leaves. Silencing SlRDR1 using virus-induced gene silencing system significantly reduced SlRDR1 expression and tomato defense against TMV but had no evident effect on SlAOX1a transcription. Conversely, silencing SlAOX1a not only effectively reduced the AOX1a transcript level, but also blocked the TMV-induced SlRDR1 expression and decreased the basal defense against TMV. Furthermore, the application of an exogenous AOX activator on empty vector-silenced control plants greatly induced the accumulation of SlRDR1 and SlAOX1a transcript and reduced TMV viral RNA accumulation, but failed to have such effects on SlRDR1-silenced plants. Moreover, RDR1-overexpressed transgenic Nicotiana benthamiana plants enhanced defense against TMV than the empty vector-transformed plants, but these effects were not affected by the exogenous AOX activator or inhibitor. These results indicate that RDR1 is involved in the AOX-mediated defense pathway against TMV infection and plays a crucial role in enhancing RNA silencing to limit virus systemic spread.
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Flavonoid ingredients of.
J. Biomed. Sci.
PUBLISHED: 09-03-2014
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BackgroundLipid accumulation is the primary evidence of non-alcoholic fatty liver disease (NAFLD). Ginkgo biloba extract (GBE) and its flavonoid ingredients (quercetin, kaempferol, and isorhamnetin) could lessen the lipid accumulation associated with up-regulation of the rate-limiting enzyme, carnitine palmitoyltransferase 1A (CPT1A), in the ß-oxidation of long-chain fatty acids. In this study, we investigated the mechanisms by which GBE and its flavonoids induced expression of CPT1A.ResultsCPT1A inhibition with RNAi resulted in triglyceride accumulation in HepG2 cells. Through deletion and mutation analysis of CPT1A¿s promoter combined with electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments, the CPT1A promoter region (¿50 to ¿5 nt) was determined to contain two putative Sp1 binding sites, namely Sp1a and Sp1b, which might act as the GBE regulation response DNA element. Sp1 might be induced to transfer from cytoplasma to nucleus to bind the promoter region of ¿50 to ¿5 nt by GBE. The regulatory effects of GBE on CPT1A were also verified on the flavonoid ingredients quercetin, kaempferol, and isorhamnetin.ConclusionSp1 was crucial in regulating CPT1A expression with GBE and its flavonoid ingredients, and the ¿50 to ¿5 nt region of CPT1A promoter played important roles in Sp1 binding.
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Sensitivity of MRI-guided near-infrared spectroscopy clinical breast exam data and its impact on diagnostic performance.
Biomed Opt Express
PUBLISHED: 09-01-2014
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In this study, data from breast MRI-guided near infrared spectroscopy (NIRS) exams delivered to 44 patients scheduled for surgical resection (ending in 16 benign and 28 malignant diagnoses) were analyzed using a spatial sensitivity metric to quantify the adequacy of the optical measurements for interrogating the tumor region of interest, as derived from the concurrent MRI scan. Along with positional sensitivity, the incorporation of spectral priors and the selection of an appropriate regularization parameter in the image reconstruction were considered, and found to influence the diagnostic accuracy of the recovered images. Once optimized, the MRI/NIRS data was able to differentiate the malignant from benign lesions through both total hemoglobin (p = 0.0037) and tissue optical index (p = 0.00019), but required the relative spatial sensitivity of the optical measurement data to each lesion to be above 1%. Spectral constraints implemented during the reconstruction were required to obtain statistically significant diagnostic information from images of H2O, lipids, and Tissue Optical Index (TOI). These results confirm the need for optical systems that have homogenous spatial coverage of the breast while still being able to accommodate the normal range of breast sizes.
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The M235T polymorphism in the angiotensinogen gene and atrial fibrillation: A meta-analysis.
J Renin Angiotensin Aldosterone Syst
PUBLISHED: 08-28-2014
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The M235T polymorphism in the angiotensinogen gene has been reported to be associated with the development of atrial fibrillation (AF).However, results from observational studies are conflicting.
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Curcumin promotes KLF5 proteasome degradation through downregulating YAP/TAZ in bladder cancer cells.
Int J Mol Sci
PUBLISHED: 08-28-2014
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KLF5 (Krüppel-like factor 5) plays critical roles in normal and cancer cell proliferation through modulating cell cycle progression. In this study, we demonstrated that curcumin targeted KLF5 by promoting its proteasome degradation, but not by inhibiting its transcription in bladder cancer cells. We also demonstrated that lentivirus-based knockdown of KLF5 inhibited cancer cell growth, while over-expression of a Flag-tagged KLF5 could partially reverse the effects of curcumin on cell growth and cyclin D1 expression. Furthermore, we found that curcumin could down-regulate the expression of Hippo pathway effectors, YAP and TAZ, which have been reported to protect KLF5 protein from degradation. Indeed, knockdown of YAP by small interfering RNA caused the attenuation of KLF5 protein, but not KLF5 mRNA, which was reversed by co-incubation with proteasome inhibitor. A xenograft assay in nude mice finally proved the potent inhibitory effects of curcumin on tumor growth and the pro-proliferative YAP/TAZ/KLF5/cyclin D1 axis. Thus, our data indicates that curcumin promotes KLF5 proteasome-dependent degradation through targeting YAP/TAZ in bladder cancer cells and also suggests the therapeutic potential of curcumin in the treatment of bladder cancer.
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Male mate recognition via cuticular hydrocarbons facilitates sexual isolation between sympatric leaf beetle sister species.
J. Insect Physiol.
PUBLISHED: 08-27-2014
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Chemical signals in insects have been documented to play an important role in mate recognition, and divergence in chemical signals can often cause sexual isolation between closely related species or populations within species. We investigated the role of cuticular hydrocarbons (CHCs), short distance chemical signals, in male mate recognition between the two sympatric elm leaf beetles, Pyrrhalta maculicollis and Pyrrhaltaaenescens. Mating experiments demonstrated that strong sexual isolation between the two species was driven by CHCs divergence. Males preferred to mate with conspecific females with intact conspecific CHCs or conspecific CHCs reapplied after removal. Males also preferred heterospecific females that were treated with conspecific CHCs. Chemical analysis showed that the CHC profiles differ significantly between species. In P. maculicollis dimethyl-branched alkanes between C29 and C35 account for the majority of the saturated alkanes while the CHC profile of P. aenescens mostly consisted of monomethyl-branched alkanes between C22 and C29. Additionally, some compounds, such as 12,18-diMeC32, 12,18-diMeC34, are unique to P. maculicollis.
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[Synthesis and in vitro cytotoxic activities of sorafenib derivatives].
Yao Xue Xue Bao
PUBLISHED: 08-26-2014
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A series of novel sorafenib analogues were designed and synthesized. The cytotoxic activities of these compounds were tested in four tumor cell lines. Some of the compounds showed potent antiproliferative activity against the tested cell lines with IC50 = 4-20 micromol x L(-1). Some compounds demonstrated competitive antiproliferative activities to sorafenib against tested cancer cell lines. Among them, compound 7c demonstrated significant inhibitory activities on ACHN, HCT116 and MDA-MB-231 cell lines with IC50 values of 9.01, 4.97, 6.61 micromol x L(-1), respectively.
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Effect of low estrogen on neurons in the preoptic area of hypothalamus of ovariectomized rats.
Acta Histochem.
PUBLISHED: 08-18-2014
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The purpose of this study was to investigate the difference in neuronal activity in the preoptic area of the hypothalamus (POAH) under low estrogen condition induced by ovariectomy. One hundred and twenty sham-operated (SHAM) and ovariectomized (OVX) rats were placed in different temperatures for 2h. Twelve rats from each group were stimulated by 4°C, 10°C, 25°C, 33°C and 38°C, respectively. c-Fos expression in the POAH was detected by immunohistochemistry. Following exposure to warm and cold stimuli, there were markedly lower c-Fos-positive cell densities in the OVX group compared with the SHAM group in the median preoptic nucleus (MnPO) at 4°C, 10°C, 33°C and 38°C, in the medial preoptic area (MPA) at 25°C and 38°C, in the ventromedial preoptic nucleus (VMPO) at 4°C, 10°C and 38°C and in the ventrolateral preoptic nucleus (VLPO) at 4°C and 38°C. Both temperature and surgery had an impact on c-Fos expression by two-way ANOVA method except in the lateral preoptic area (LPO). c-Fos expression differed within different nuclei of the two groups in the same and different temperature stimuli. This indicated that the temperature-sensitive nuclei in the POAH exhibited lower and different activities during temperature stimuli following ovariectomy, which possibly resulted in abnormal thermoregulation and menopausal symptoms.
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Hydroxypropyl-?-cyclodextrin grafted polyethyleneimine used as transdermal penetration enhancer of diclofenac sodium.
Int J Pharm
PUBLISHED: 08-17-2014
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The objective of this investigation was to develop a novel cationic polymer, hydroxypropyl-?-cyclodextrin grafted polyethyleneimine (HP-?-CD-PEI1800), as a penetration enhancer, and evaluate its viability on improving transdermal delivery of diclofenac sodium. In this study, HP-?-CD-PEI1800 was characterized by (1)H NMR and DSC methods, respectively. The hydrophilic drug diclofenac sodium was chosen as model drug, and the transdermal permeation enhancement of HP-?-CD-PEI1800 was estimated in vitro by using Franz diffusion cells fitted with mouse dorsal skins, the in vivo kinetics of diclofenac sodium was analyzed by high-performance liquid chromatography (HPLC). The cumulative drug content deposited in epidermis and dermis was measured at the pre-determined time point of 3, 6, and 9h, and the permeation profile was significantly higher than that of the control groups. In addition, the cytotoxicity and skin irritation of enhancer was evaluated by MTT assay and histological examination, respectively, and the results indicated that the polymer we prepared were non-toxic and non-irritant after exposure to skins. All the results suggested that HP-?-CD-PEI1800 could be a safe and efficient penetration enhancer of diclofenac sodium.
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Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells.
Folia Neuropathol
PUBLISHED: 08-15-2014
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Ciliary neurotrophic factor (CNTF) is a neurocytokine, which could promote survival and/or differentiation in many cell types. In this study, the biological effects of CNTF on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells and the underlying molecular mechanism of this effect were investigated for the first time. The results showed that RA was able to increase cells susceptibility to CNTF via regulating the expression levels of CNTF receptors. A further study revealed that CNTF could induce phosphorylation of STAT3, Akt and ERK1/2 in RA-predifferentiated SH-SY5Y neuroblastoma cells, while the promoting activity of CNTF on survival and neurite growth of cells was attenuated by co-treatment with JAK2 inhibitor AG490 (25 ?M), STAT3 inhibitor Curcumin (50 ?M), PI3K inhibitor LY-294002 (50 µM), but not by co-treatment with MEK inhibitor PD98059 (50 ?M). These findings suggested that JAK2/STAT3, as well as PI3K/Akt, play important roles in mediating the survival and neurite growth response of RA-predifferentiated cells to CNTF. Our study may be useful to further understand the functional role of CNTF and offer a convenient model to explore the therapeutic potential of CNTF in neurodegenerative diseases.
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Anti-peroxynitrite treatment ameliorated vasorelaxation of resistance arteries in aging rats: involvement with NO-sGC-cGKs pathway.
PLoS ONE
PUBLISHED: 08-12-2014
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Declined vasorelaxation function in aging resistance arteries is responsible for aging-related multiple organ dysfunctions. The aim of the present study is to explore the role of peroxynitrite (ONOO-) in aging resistance arterial vasorelaxation dysfunction and the possible mechanism. In the present study, young (3-4 months olds) and aging (20 months olds) male SD rats were randomized to receive vehicle (Saline) or FeTMPyP (ONOO- scavenger) for 2 weeks. The vasorelaxation of resistance arteries was determined in vitro; NOx level was tested by a colorimetric assay; the expression of nitrotyrosine (NT), soluble Guanylate Cyclase (sGC), vasodilator-stimulated phosphoprotein (VASP), phosphorylated VASP (P-VASP) and cGMP in resistance arteries were detected by immunohistochemical staining. In the present study, endothelium-dependent dilation in aging resistance arteries was lower than in those from young rats (young vs. aging: 68.0% ± 4.5% vs. 50.4% ± 2.9%, P<0.01). And the endothelium-independent dilation remained constant. Compared with young rats, aging increased nitrative stress in resistance arteries, evidenced by elevated NOx production in serum (5.3 ± 1.0 nmol/ml vs. 3.3 ± 1.4 nmol/ml, P<0.05) and increased NT expression (P<0.05). ONOO- was responsible for the vasorelaxation dysfunction, evidenced by normalized vasorelaxation after inhibit ONOO- or its sources (P<0.05) and suppressed NT expression after FeTMPyP treatment (P<0.05). The expression of sGC was not significantly different between young and aging resistance arteries, but the cGMP level and P-VASP/VASP ratio (biochemical marker of NO-sGC-cGKs signaling) decreased, which was reversed by FeTMPyP treatment in vivo (P<0.05). The present study suggested that ONOO- mediated the decline of endothelium-dependent vasorelaxation of aging resistance arteries by induction of the NO-sGC-cGKs pathway dysfunction.
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Redox-based selective fluorometric detection of homocysteine.
Chem. Commun. (Camb.)
PUBLISHED: 08-07-2014
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Plasma homocysteine (Hcy) is an important risk factor for various diseases. A novel redox-sensitive fluorescent probe is developed for the selective detection of Hcy. A linear calibration curve has been obtained in buffer and plasma for the quantitative determination of Hcy in such media.
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An update to the taxonomy of the genus Gastroserica Brenske (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 07-17-2014
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Based on the examination of newly collected material and additional specimens housed in Chinese collections, our knowledge of Gastroserica Brenske, 1897, is expanded. Here, seven new species are described, including habitus photographs and illustrations of the male genitalia: Gastroserica haoyui sp. n. (China: Zhejiang Prov.), G. fengduana sp. n. (China: Sichuan Prov.), G. wenzhui sp. n. (China: Guangxi Prov.), G. damingshanica sp. n. (from China: Guangxi Prov.), G. jinxiuensis sp. n. (China: Guangxi Prov.), G. liboensis sp. n. (China: Yunnan Prov.) and G. carolusi sp. n. (Laos). Additionally, we provide a distribution map of the new taxa and new distribution records of the known taxa.
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Determination of the Stereochemistry of the Aggregation Pheromone of Harlequin Bug, Murgantia histrionica.
J. Chem. Ecol.
PUBLISHED: 07-16-2014
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Preparation of a complete stereoisomeric library of 1,10-bisaboladien-3-ols and selected 10,11-epoxy-1-bisabolen-3-ols was pivotal for the identification of the aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys. Herein, we describe syntheses of the remaining 10,11-epoxy-1-bisabolen-3-ols, and provide additional evidence on the assignment of relative and absolute configurations of these compounds by single-crystal X-ray crystallography of an intermediate, (3S,6R,7R,10S)-1-bisabolen-3,10,11-triol. To demonstrate the utility of this stereoisomeric library, we revisited the aggregation pheromone of the harlequin bug, Murgantia histrionica, and showed that the male-produced pheromone consists of two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol. Employment of eight cis-10,11-epoxy-1-bisabolen-3-ol stereoisomeric standards, two enantioselective GC columns, and NMR spectroscopy enabled the identification of these compounds as (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol and (3S,6S,7R,10R)-10,11-epoxy-1-bisabolen-3-ol, which are produced by M. histrionica males in 1.4:1 ratio.
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Plasma Interleukin-10: A Likely Predictive Marker for Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.
Hepat Mon
PUBLISHED: 07-14-2014
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The pathogenesis of HBV-related acute-on-chronic liver failure (HBV-ACLF) is mainly based on a heightened immune-inflammatory reaction; however, the intimate underlying mechanism remains unclear.
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Impact of individual extracellular proteases on Staphylococcus aureus biofilm formation in diverse clinical isolates and their isogenic sarA mutants.
Microbiologyopen
PUBLISHED: 07-08-2014
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We demonstrate that the purified Staphylococcus aureus extracellular proteases aureolysin, ScpA, SspA, and SspB limit biofilm formation, with aureolysin having the greatest impact. Using protease-deficient derivatives of LAC, we confirmed that this is due to the individual proteases themselves. Purified aureolysin, and to a lesser extent ScpA and SspB, also promoted dispersal of an established biofilm. Mutation of the genes encoding these proteases also only partially restored biofilm formation in an FPR3757 sarA mutant and had little impact on restoring virulence in a murine bacteremia model. In contrast, eliminating the production of all of these proteases fully restored both biofilm formation and virulence in a sarA mutant generated in the closely related USA300 strain LAC. These results confirm an important role for multiple extracellular proteases in S. aureus pathogenesis and the importance of sarA in repressing their production. Moreover, purified aureolysin limited biofilm formation in 14 of 15 methicillin-resistant isolates and 11 of 15 methicillin-susceptible isolates, while dispersin B had little impact in UAMS-1, LAC, or 29 of 30 contemporary isolates of S. aureus. This suggests that the role of sarA and its impact on protease production is important in diverse strains of S. aureus irrespective of their methicillin resistance status.
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Chronic alcohol consumption from adolescence to adulthood in mice--hypothalamic gene expression changes in insulin-signaling pathway.
Alcohol
PUBLISHED: 07-05-2014
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Adolescence is a developmental stage vulnerable to alcohol drinking-related problems, and alcohol exposure during adolescence may lead to long-lasting consequences. The hypothalamus is a key brain region for food and water intake regulation as well as weight control, and is one of the alcohol-sensitive brain regions. However, it is not known what the alcohol effect is on the hypothalamus following adolescent alcohol intake, chronically over adolescent development, at moderate levels. We employed a model of chronic moderate alcohol intake from adolescence to adulthood in mice, and analyzed the effect of alcohol on growth and weight gain, as well as hypothalamic gene expression patterns. The results indicated that chronic alcohol consumption during adolescence, even at moderate levels, led to both a reduction in weight gain in mice, and considerable gene expression changes in the hypothalamus. Pathway analysis and real-time PCR identified the type II diabetes mellitus and the insulin-signaling pathways as being the hypothalamic pathways affected by chronic alcohol. Our findings from the mouse alcohol consumption study therefore serve as a potential warning against alcohol consumption during adolescence, such as in teens and college students.
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Characterization of local delivery with amphotericin B and vancomycin from modified chitosan sponges and functional biofilm prevention evaluation.
J. Orthop. Res.
PUBLISHED: 06-30-2014
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Polymicrobial musculoskeletal wound infections are troublesome complications and can be difficult to treat when caused by invasive fungi or bacteria. However, few local antifungal delivery systems have been studied. Chitosan and polyethylene glycol (PEG) sponge local antifungal delivery systems have been developed for adjunctive therapy to reduce musculoskeletal wound contamination. This study evaluated the effects of blending PEG, at 6,000 or 8,000?g/mol, with chitosan in sponge form on in vitro amphotericin B and vancomycin elution, eluate activity, cytocompatibility, and in vivo prevention of a bacterial biofilm. Blended chitosan sponges released both amphotericin B and vancomycin in vitro. All tested amphotericin B eluates remained active against Candida albicans, and vancomycin eluates from blended sponges maintained activity against Staphylococcus aureus. Amphotericin B eluates obtained after one hour from blended sponges elicited 62-95% losses in fibroblast viability, but three hour eluates only caused 22-60% decreases in viability. In a Staphylococcus aureus infected mouse catheter biofilm prevention model, vancomycin loaded chitosan/PEG 6000 sponge cleared bacteria from 100% of the catheters, with reduced clearance rate observed in other sponges. These results indicate that the chitosan/PEG blended sponges have potential for local antifungal and/or antibiotic combination delivery as an adjunctive therapy to prevent wound infections. This article is protected by copyright. All rights reserved.
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Genomewide analysis of rat periaqueductal gray-dorsal horn reveals time-, region- and frequency-specific mRNA expression changes in response to electroacupuncture stimulation.
Sci Rep
PUBLISHED: 06-30-2014
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Electroacupuncture (EA) has been widely applied for illness prevention, treatment or rehabilitation in the clinic, especially for pain management. However, the molecular events that induce these changes remain largely uncharacterized. The periaqueductal gray (PAG) and the spinal dorsal horn (DH) have been verified as two critical regions in the response to EA stimulation in EA analgesia. In this study, a genetic screen was conducted to delineate the gene expression profile in the PAG-DH regions of rats to explore the molecular events of the analgesic effect induced by low-frequency (2-Hz) and high-frequency (100-Hz) EAs. Microarray analysis at two different time points after EA stimulation revealed time-, region- and frequency-specific gene expression changes. These expression differences suggested that modulation of neural-immune interaction in the central nervous system played an important role during EA analgesia. Furthermore, low-frequency EA could regulate gene expression to a greater degree than high-frequency EA. Altogether, the present study offers, for the first time, a characterized transcriptional response pattern in the PAG-DH regions followed by EA stimulation and, thus, provides a solid experimental framework for future in-depth analysis of the mechanisms underlying EA-induced effects.
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Never in mitosis gene A-related kinase 6 promotes cell proliferation of hepatocellular carcinoma via cyclin B modulation.
Oncol Lett
PUBLISHED: 06-30-2014
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Never in mitosis gene A-related kinase (Nek) 6 is a recently identified Nek that is required for mitotic cell cycle progression; however, the role and mechanism of Nek6 activity during hepatocarcinogenesis is not well known. The aim of this study was to investigate the potential roles and internal mechanism of Nek6 in hepatocellular carcinoma (HCC) development. In the present study, Nek6 was found to be overexpressed in HCC samples and cell lines by florescent real-time quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. Furthermore, it was evidenced to contribute to oncogenesis and progression. The ectopic overexpression of Nek6 promoted cell proliferation and colony formation, whereas gene silencing of Nek6 inhibited these phenotypes, as documented in Huh7, PLC/PRF/5, Hep3B and HepG2 HCC cell lines. Mechanistic analyses indicated that Nek6 regulates the transcription of cyclin B through cdc2 activation, and promotes the accumulation of G0/G1-phase cells. In conclusion, the findings of the current study suggested that Nek6 contributes to the oncogenic potential of HCC, and may present as a potential therapeutic target in this disease.
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Discovery of the aggregation pheromone of the brown marmorated stink bug (Halyomorpha halys) through the creation of stereoisomeric libraries of 1-bisabolen-3-ols.
J. Nat. Prod.
PUBLISHED: 06-25-2014
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We describe a novel and straightforward route to all stereoisomers of 1,10-bisaboladien-3-ol and 10,11-epoxy-1-bisabolen-3-ol via the rhodium-catalyzed asymmetric addition of trimethylaluminum to diastereomeric mixtures of cyclohex-2-enones 1 and 2. The detailed stereoisomeric structures of many natural sesquiterpenes with the bisabolane skeleton were previously unknown because of the absence of stereoselective syntheses of individual stereoisomers. Several of the bisabolenols are pheromones of economically important pentatomid bug species. Single-crystal X-ray crystallography of underivatized triol 13 provided unequivocal proof of the relative and absolute configurations. Two of the epoxides, (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (4), were identified as the main components of a male-produced aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys, using GC analyses on enantioselective columns. Both compounds attracted female, male, and nymphal H. halys in field trials. Moreover, mixtures of stereoisomers containing epoxides 3 and 4 were also attractive to H. halys, signifying that the presence of additional stereoisomers did not hinder attraction of H. halys and relatively inexpensive mixtures can be used in monitoring, as well as control strategies. H. halys is a polyphagous invasive species in the U.S. and Europe that causes severe injury to fruit, vegetables, and field crops and is also a serious nuisance pest.
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Antifouling potential of the marine microalga Dunaliella salina.
World J. Microbiol. Biotechnol.
PUBLISHED: 06-23-2014
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Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC50 value against Skeletonema costatum was 58.9 ?g ml(-1). The isolated purified extract was tested for antifouling activity, the EC 50 value against S. costatum was 21.2 ?g ml(-1), and the LC50 against Balanus amphitrite larvae was 18.8 ?g ml(-1). Subsequently, both UHR-TOF-MS and GC-MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.
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High-level expression of LMW-GS and ?-gliadin genes promoted by the expressed tag sequence of 5' end in Escherichia coli.
Protein Expr. Purif.
PUBLISHED: 06-19-2014
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Wheat storage protein genes, especially low molecular weight glutenin subunit (LMW-GS) and gliadin genes are difficult to be expressed in Escherichiacoli, mainly due to the presence of highly repetitive sequences. In order to establish a high efficiency expression system for these genes, five different expression plasmids combining with 9 genes, viz. 6 LMW-GS and 3 ?-gliadin genes isolated from common wheat and related species, were studied for heterologous expression in E. coli. In this study, when an expressed tag sequence encoding signal peptide, His-S or GST-tag was fused to the 5' end of LMW-GS or gliadin gene as the leading sequence, all recombination genes could be stably expressed at a high level. On the contrast, as expected, the inserted genes encoding mature protein failed without an expressed tag sequence. This result indicated that using expressed tag sequences as leading sequences could promote LMW-GS and gliadin genes to be well expressed in E. coli. Further transcriptional analysis by quantitative real-time PCR (qRT-PCR) showed transcription levels of recombination genes (e.g. GST-Glutenin, His-S-Glutenin and SP(?)-His-Glutenin) were 4-fold to 33-fold higher than those of the LMW-GS genes, which suggested these expressed tag sequences might play an important role in stimulating transcription. The possible molecular mechanism under this phenomenon was discussed.
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Broadband and broad-angle low-scattering metasurface based on hybrid optimization algorithm.
Sci Rep
PUBLISHED: 06-10-2014
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A broadband and broad-angle low-scattering metasurface is designed, fabricated, and characterized. Based on the optimization algorithm and far-field scattering pattern analysis, we propose a rapid and efficient method to design metasurfaces, which avoids the large amount of time-consuming electromagnetic simulations. Full-wave simulation and measurement results show that the proposed metasurface is insensitive to the polarization of incident waves, and presents good scattering-reduction properties for oblique incident waves.
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Structural characterization of (1?2)-?-xylose-(1?3)-?-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with ?-arabinofuranosidase and ?-endo-xylanase.
Carbohydr. Res.
PUBLISHED: 05-22-2014
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Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation and to reduce inhibitory effects xylooligomers may have on cellulolytic glycoside hydrolase enzymes. To identify structural features of switchgrass xylan that are not distinguishable by mass spectrometry alone, a ?-arabinofuranosidase enzyme was used to remove the arabinose side chains from alkali-extracted switchgrass xylan from three cultivars with simultaneous hydrolysis by ?-endo-xylanase to enrich for oligosaccharide products with extended branching. The two most abundant enzymatic digestion products were separated and characterized by LC-MS(n), linkage analysis, and NMR. These two oligosaccharides were present in all three switchgrass cultivars and found to contain (1?2)-?-xylose-(1?3)-?-arabinose side chains, a linkage not previously reported in switchgrass.
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Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis.
J Biomater Appl
PUBLISHED: 05-21-2014
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We demonstrate that coating calcium sulfate with deacetylated chitosan enhances the elution profile of daptomycin by prolonging the period during which high concentrations of antibiotic are released. Coatings reduced initial bolus release of daptomycin by a factor of 10 to approximately 1000?µg/ml, and levels remained above 100?µg/ml for up to 10 days. Chitosan-coated and uncoated calcium sulfate implants with and without 15% daptomycin were evaluated in an experimental model of staphylococcal osteomyelitis through bacteriology scores, radiology, histopathology, and Gram staining. Significant reduction in bacteriology scores was observed for implants containing daptomycin and coated with chitosan compared with all the other groups. We confirm that the use of chitosan-coated calcium sulfate beads for local antibiotic delivery can be correlated with an improved therapeutic outcome following surgical debridement in the treatment of chronic osteomyelitis.
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Efficacy and acceptability of atypical antipsychotics for the treatment of post-traumatic stress disorder: a meta-analysis of randomized, double-blind, placebo-controlled clinical trials.
Psychiatry Res
PUBLISHED: 05-05-2014
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As some evidences demonstrated that atypical antipsychotics (AA) may be efficacious in treating post-traumatic stress disorder (PTSD), we preformed a meta-analysis of randomized, double-blind, placebo-controlled clinical trials (RCTs) of AAs for the treatment of PTSD. Two hundred and fifty one papers were searched and screened. Eight RCTs met the inclusion criteria. AAs may be superior to placebo in the treatment of PTSD, as indicated by the changes in Clinician Administered PTSD Scale (CAPS) total scores (weighted mean differences (WMD)=-5.89, 95% confidence interval (CI) [-9.21, -2.56], P=0.0005) and also in CAPS subscale intrusion (WMD=-2.58, 95% CI[-3.83, -1.33], P<0.0001 ) and subscale hyperarousal (WMD=-2.94, 95% CI[-5.45, -0.43], P=0.02). The acceptability measured by dropout rates between AAs and placebo showed no statistical difference (OR=1.24, 95%CI [0.78, 1.97], P=0.36). PTSD symptom cluster, especially in intrusion and hyperarousal. However, we should be careful to generalize the conclusion because of the small number of included trails. We expect more RCTs will be done in the future so as to clarify the specific value of AAs for PTSD.
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Impact of the functional status of saeRS on in vivo phenotypes of Staphylococcus aureus?sarA mutants.
Mol. Microbiol.
PUBLISHED: 04-25-2014
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We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS(C)) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.
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The combination of a nuclear HMGB1-positive and HMGB2-negative expression is potentially associated with a shortened survival in patients with pancreatic ductal adenocarcinoma.
Tumour Biol.
PUBLISHED: 04-18-2014
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High-mobility group box (HMGB) proteins are ubiquitous, abundant nuclear non-histone chromosomal proteins that play a critical role in binding to distorted DNA structures and subsequently regulating DNA transcription, replication, repair, and recombination. Both HMGB1 and HMGB2 exhibit a high expression in several human cancers and are closely associated with tumor progression and a poor prognosis. However, the expression patterns of these molecules in pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. As most cases of postoperative relapse of PDAC occur within the first 2 years, the clinical significance of accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical HMGB1 and HMGB2 expression and the clinicopathological characteristics and prognosis using 62 paraffin-embedded tumor samples obtained from patients with surgically resected PDAC. The HMGB1/2 expression was considered to be positive when 10 % or more of the cancer cells showed positive nuclear, not merely cytoplasmic, staining. Consequently, the expression of HMGB1/2 was observed in 54 (87.1 %) and 31 (50.0 %) patients, respectively. Unexpectedly, a positive HMGB1 expression was found to have a significantly close relationship with a negative HMGB2 expression. The univariate and multivariate analyses demonstrated that the patients with a HMGB1+ and HMGB2- status had markedly lower disease-specific survival rates, especially within the first 2 years postoperatively, whereas those with a HMGB1+ status alone did not. Therefore, the combination of a HMGB1+ and HMGB2- expression potentially predicts a poor prognosis in patients with PDAC, and these new biomarkers may be useful parameters for clinical management in the early postoperative phase.
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Nitric oxide exerts protective effects against bleomycin-induced pulmonary fibrosis in mice.
Respir. Res.
PUBLISHED: 03-04-2014
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Increased expression of nitric oxide synthase (NOS) and an increase in plasma nitrite plus nitrate (NOx) have been reported in patients with pulmonary fibrosis, suggesting that nitric oxide (NO) plays an important role in its development. However, the roles of the entire NO and NOS system in the pathogenesis of pulmonary fibrosis still remain to be fully elucidated. The aim of the present study is to clarify the roles of NO and the NOS system in pulmonary fibrosis by using the mice lacking all three NOS isoforms.
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Assortative mating between two sympatric closely-related specialists: inferred from molecular phylogenetic analysis and behavioral data.
Sci Rep
PUBLISHED: 02-18-2014
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Host plant shifting of phytophagous insects can lead to the formation of host associated differentiation and ultimately speciation. In some cases, host plant specificity alone acts as a nearly complete pre-mating isolating barrier among insect populations. We here test whether effective pre-mating isolation and host-independent behavioral isolation have evolved under the condition of extreme host specilization using two sympatric flea beetles with incomplete post-mating isolation under laboratory conditions. Phylogenetic analysis and coalescent simulation results showed that there is a limited interspecific gene flow, indicating effctive isolation between these species. Three types of mating tests in the absence of host plant cues showed that strong host-independent behavioral isolation has evolved between them. We conclude that almost perfect assortative mating between these two extreme host specialists results from a combination of reduced encounter rates due to differential host preference and strong sexual isolation.
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Red emissive cross-linked chitosan and their nanoparticles for imaging the nucleoli of living cells.
Carbohydr Polym
PUBLISHED: 02-11-2014
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Biocompatible glutaraldehyde-cross-linked chitosan with new red fluorescence were prepared for the first time and were shaped into nanoparticles via inverse-microemulsion method. They could luminesce at ca. 670 nm either as powders and nanoparticles or in real and gelling solutions or suspensions, having a lifetime of 1.353 ns and a quantum yield of 0.08 in solution or 0.01 in solid state. The new-formed pyridinium structures and the intramolecular charge transfer effect are considered to be responsible for the new red emission, which have been proved by FTIR, (13)C NMR, and some calculation using Gaussian 09, respectively. Strikingly, they are quite inert and anti-photobleaching, with only <3% loss of fluorescent intensity per minute in average under a continuous laser illumination at 633 nm and 50 ?W. Especially, their nanoparticles (5.6 nm) could enter into the negative nucleoli of living HeLa cells with low cytotoxicity for high contrast imaging inspections.
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Hypersensitivity to aurora kinase inhibitors in cells resistant against platinum- containing anticancer agents.
Anticancer Agents Med Chem
PUBLISHED: 02-03-2014
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The aurora kinases are serine/threonine kinases that are essential for mitosis and contribute to tumorigenesis. Therefore, aurora kinases hold promise for molecularly targeted therapy. In the present study, we demonstrated that aurora B kinase (AURKB) is overexpressed in both cisplatin- and oxaliplatin-resistant cells. Downregulation of AURKB sensitized cells to both cisplatin and oxaliplatin, but not to paclitaxel, 5-FU or hydrogen peroxide. Interestingly, we found that both cisplatin- and oxaliplatin-resistant cells were hypersensitive to the AURKB specific inhibitors, AZD1152 HQPA and ZM447439, suggesting that both cisplatin- and oxaliplatinresistant cells develop an addiction to AURKB. These data provide evidence that aurora kinase inhibitors can overcome both cisplatin and oxaliplatin resistance. Therefore, AURKB inhibitors could offer potential benefits if used after first-line platinum-based chemotherapy.
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Glucansucrase acceptor reactions with d-mannose.
Carbohydr. Res.
PUBLISHED: 01-10-2014
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The main acceptor product of glucansucrases with d-mannose has not previously been identified. We used glucansucrases that form water-insoluble ?-d-glucans to produce increased yields of acceptor products from d-mannose, and identified the major product as 6-O-?-d-glucopyranosyl-d-mannose. Glucansucrases that synthesize insoluble ?-d-glucans produced higher yields of the disaccharide compared to typical dextransucrases.
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Depletion of apoptosis signal-regulating kinase 1 prevents bile duct ligation-induced necroinflammation and subsequent peribiliary fibrosis.
Am. J. Pathol.
PUBLISHED: 01-09-2014
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Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase (MAP3K), is ubiquitously expressed and situated in an important upstream position of many signal transduction pathways. ASK1 plays a pivotal role in stressor-induced cell survival and inflammatory reactions. To ascertain the regulatory functions of ASK1 in bile duct ligation (BDL)-induced liver injury, we examined the net effects of ASK1 depletion on hepatic necroinflammation and/or fibrosis. We subjected C57BL/6 wild-type (WT) or ASK1-deficient (ASK1(-/-)) mice to sham or BDL surgery for 14 days. In day 3 BDL animals, ASK1(-/-) mice had significantly fewer bile infarcts along with more reduced interlobular or portal inflammatory infiltrate of various immune cells, including neutrophils, compared with WT mice in which ASK1 expression was markedly activated. Morphologically apoptotic hepatocytes or cholangiocytes were negligible in both the sham and BDL animals. In contrast, ASK1(-/-) mice had significantly less proliferating activity of not only hepatocytes but also large cholangiocytes than WT mice. Day 14 BDL ASK1(-/-) mice manifested potential antifibrogenic aspects of ASK1 deficiency, characterized by significantly fewer activated peribiliary fibrogenic cells and peribiliary fibrosis. These observations indicate that ASK1-mediated hepatic necroinflammation and proliferation, but not apoptosis, are closely linked to liver fibrosis and fibrogenesis. A specific ASK1 pathway blocker or inhibitor might offer a therapeutic strategy against human cholestatic diseases.
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Marine Hydroquinone Zonarol Prevents Inflammation and Apoptosis in Dextran Sulfate Sodium-Induced Mice Ulcerative Colitis.
PLoS ONE
PUBLISHED: 01-01-2014
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We previously identified an anti-inflammatory compound, zonarol, a hydroquinone isolated from the brown algae Dictyopteris undulata as a marine natural product. To ascertain the in vivo functions of zonarol, we examined the pharmacological effects of zonarol administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of ulcerative colitis (UC). Our goal is to establish a safe and effective cure for inflammatory bowel disease (IBD) using zonarol.
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Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp.
PLoS ONE
PUBLISHED: 01-01-2014
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The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n?=?11) and Aspergillus (n?=?3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ?6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (?2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.
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A taxonomic review on the species of Tetraserica Ahrens, 2004, of China (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 01-01-2014
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A review on the Chinese species of Tetraserica Ahrens, 2004, is presented. The lectotype of Tetrasericatonkinensis (Moser, 1908), comb. n. is designated. Twenty-nine new Tetraserica species are described from China and adjacent regions: Tetrasericaanhuaensis sp. n., Tetrasericachangjiangensis sp. n., Tetrasericachangshouensis sp. n., Tetrasericadamaidiensis sp. n., Tetrasericadaqingshanica sp. n., Tetrasericafikaceki sp. n., Tetrasericagraciliforceps sp. n., Tetrasericajinghongensis sp. n., Tetrasericaleishanica sp. n., Tetrasericaliangheensis sp. n., Tetrasericalinaoshanica sp. n., Tetrasericalongipenis sp. n., Tetrasericalongzhouensis sp. n., Tetrasericamaoershanensis sp. n., Tetrasericamengeana sp. n., Tetrasericamenglongensis sp. n., Tetrasericapingjiangensis sp. n., Tetrasericaruiliana sp. n., Tetrasericaruiliensis sp. n., Tetrasericasculptilis sp. n., Tetrasericashangsiensis sp. n., Tetrasericashunbiensis sp. n., Tetrasericasigulianshanica sp. n., Tetrasericatianchiensis sp. n., Tetrasericawandingensis sp. n., Tetrasericawangtongensis sp. n., Tetrasericaxichouensis sp. n., Tetrasericayaoanica sp. n., Tetrasericayaoquensis sp. n. A key to the Chinese Tetraserica species is given, species distribution as well as the habitus and male genitalia of all species are illustrated.
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A taxonomic revision of the Neoserica(sensu lato)calva group (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 01-01-2014
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The species of the Neoserica(sensu lato)calva group are revised. Neosericacalva Frey, 1972, comb. n. is redescribed. Thirteen new species are described from China and South Korea: Neosericaailaoshanica sp. n., Neosericaanonyma sp. n., Neosericacalvoides sp. n., Neosericagulinqingensis sp. n., Neosericakoelkebecki sp. n., Neosericaliangi sp. n., Neosericaluxiensis sp. n., Neosericamenghaiensis sp. n., Neosericamengi sp. n., Neosericataipingensis sp. n., Neosericazheijangensis sp. n., Neosericazhibenshanica sp. n., and Neosericazongyuani sp. n. A key to Sericini genera with multilamellate antenna and species groups of Neoserica of mainland Asia as well as a key to species of the Neosericacalva group are provided. A map of species distribution is given, habitus and male genitalia are illustrated.
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Clinical response and hospital costs associated with the empirical use of vancomycin and linezolid for hospital-acquired pneumonia in a Chinese tertiary care hospital: a retrospective cohort study.
Clinicoecon Outcomes Res
PUBLISHED: 01-01-2014
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To evaluate clinical outcomes and allocation of hospital costs associated with empirical use of vancomycin or linezolid for hospital-acquired pneumonia (HAP) in the People's Republic of China.
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Monitoring urban greenness dynamics using multiple endmember spectral mixture analysis.
PLoS ONE
PUBLISHED: 01-01-2014
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Urban greenness is increasingly recognized as an essential constituent of the urban environment and can provide a range of services and enhance residents' quality of life. Understanding the pattern of urban greenness and exploring its spatiotemporal dynamics would contribute valuable information for urban planning. In this paper, we investigated the pattern of urban greenness in Hangzhou, China, over the past two decades using time series Landsat-5 TM data obtained in 1990, 2002, and 2010. Multiple endmember spectral mixture analysis was used to derive vegetation cover fractions at the subpixel level. An RGB-vegetation fraction model, change intensity analysis and the concentric technique were integrated to reveal the detailed, spatial characteristics and the overall pattern of change in the vegetation cover fraction. Our results demonstrated the ability of multiple endmember spectral mixture analysis to accurately model the vegetation cover fraction in pixels despite the complex spectral confusion of different land cover types. The integration of multiple techniques revealed various changing patterns in urban greenness in this region. The overall vegetation cover has exhibited a drastic decrease over the past two decades, while no significant change occurred in the scenic spots that were studied. Meanwhile, a remarkable recovery of greenness was observed in the existing urban area. The increasing coverage of small green patches has played a vital role in the recovery of urban greenness. These changing patterns were more obvious during the period from 2002 to 2010 than from 1990 to 2002, and they revealed the combined effects of rapid urbanization and greening policies. This work demonstrates the usefulness of time series of vegetation cover fractions for conducting accurate and in-depth studies of the long-term trajectories of urban greenness to obtain meaningful information for sustainable urban development.
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A taxonomic revision of the Neoserica (sensu lato) pilosula group (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 01-01-2014
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Nine new species of the Neoserica (sensu lato) pilosula Moser, 1915, group are described from China: Neoserica curvipenis sp. n., N. emeishanensis sp. n., N. lincangensis sp. n., N. ludingensis sp. n., N. lushuiana sp. n., N. rangshuiensis sp. n., N. shennongjiaensis sp. n., N. tianeana sp. n., and N. weibaoshanica sp. n. The lectotype of Neoserica pilosula Moser, 1915, is designated. Habitus and male genitalia are illustrated, a key to the species of the group and a map of species distribution are given.
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A taxonomic review of the Neoserica (sensu lato) abnormis group (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 01-01-2014
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The present paper revises the species belonging to the Neoserica (sensu lato) abnormis group, so far known only with two nominal species. Twenty new species are herein described from Indochina and southern China: N. abnormoides sp. n. (Vietnam, China), N. allolaotica sp. n., N. namthaensis sp. n., N. simplicissima sp. n. (Laos), N. thailandensis sp. n. (Thailand), N. alloputaoana sp. n., N. kanphantensis sp. n., N. natmatoungensis sp. n., N. putaoana sp. n., N. taunggyiana sp. n. (Myanmar), N. lamellosa sp. n., N. tonkinea sp. n. (Vietnam), N. bairailingshanica sp. n., N. euyunnanica sp. n., N. huangi sp. n., N. jiangxiensis sp. n., N. trifida sp. n., N. yaoi sp. n., N. yingjiangensis sp. n. (China), N. cardamomensis sp. n. (Indochina and southern China). One new combination is established: Neoserica ponderosa Arrow, 1946, comb. n. The lectotypes of Neoserica abnormis Moser, 1908 and the taxonomically uncertain N. inclinata Brenske, 1898, which very likely also belongs to this species group, are designated herein. A key to the species and to species groups is given, the genitalia of all species including their habitus are illustrated. Maps of species distribution are included.
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Effects of Winter Cover Crops Straws Incorporation on CH4 and N2O Emission from Double-Cropping Paddy Fields in Southern China.
PLoS ONE
PUBLISHED: 01-01-2014
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Residue management in cropping systems is believed to improve soil quality. However, the effects of residue management on methane (CH4) and nitrous oxide (N2O) emissions from paddy field in Southern China have not been well researched. The emissions of CH4 and N2O were investigated in double cropping rice (Oryza sativa L.) systems with straw returning of different winter cover crops by using the static chamber-gas chromatography technique. A randomized block experiment with three replications was established in 2004 in Hunan Province, China, including rice-rice-ryegrass (Lolium multiflorum L.) (Ry-R-R), rice-rice-Chinese milk vetch (Astragalus sinicus L.) (Mv-R-R) and rice-rice with winter fallow (Fa-R-R). The results showed that straw returning of winter crops significantly increased the CH4 emission during both rice growing seasons when compared with Fa-R-R. Ry-R-R plots had the largest CH4 emissions during the early rice growing season with 14.235 and 15.906 g m-2 in 2012 and 2013, respectively, when Ry-R-R plots had the largest CH4 emission during the later rice growing season with 35.673 and 38.606 g m-2 in 2012 and 2013, respectively. The Ry-R-R and Mv-R-R also had larger N2O emissions than Fa-R-R in both rice seasons. When compared to Fa-R-R, total N2O emissions in the early rice growing season were increased by 0.05 g m-2 in Ry-R-R and 0.063 g m-2 in Mv-R-R in 2012, and by 0.058 g m-2 in Ry-R-R and 0.068 g m-2 in Mv-R-R in 2013, respectively. Similar result were obtained in the late rice growing season, and the total N2O emissions were increased by 0.104 g m-2 in Ry-R-R and 0.073 g m-2 in Mv-R-R in 2012, and by 0.108 g m-2 in Ry-R-R and 0.076 g m-2 in Mv-R-R in 2013, respectively. The global warming potentials (GWPs) from paddy fields were ranked as Ry-R-R>Mv-R-R>Fa-R-R. As a result, straw returning of winter cover crops has significant effects on increase of CH4 and N2O emission from paddy field in double cropping rice system.
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Role of WNT10A-expressing kidney fibroblasts in acute interstitial nephritis.
PLoS ONE
PUBLISHED: 01-01-2014
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WNT signaling mediates various physiological and pathological processes. We previously showed that WNT10A is a novel angio/stromagenic factor involved in such processes as tumor growth, wound healing and tissue fibrosis. In this study, we investigated the role of WNT10A in promoting the fibrosis that is central to the pathology of acute interstitial nephritis (AIN). We initially asked whether there is an association between kidney function (estimated glomerular filtration rate; eGFR) and WNT10A expression using kidney biopsies from 20 patients with AIN. Interestingly, patients with WNT10A expression had significantly lower eGFR than WNT10A-negative patients. However, changes in kidney function were not related to the level of expression of other WNT family members. Furthermore, there was positive correlation between WNT10A and ?-SMA expression. We next investigated the involvement of WNT10A in kidney fibrosis processes using COS1 cells, a kidney fibroblast cell line. WNT10A overexpression increased the level of expression of fibronectin and peroxiredoxin 5. Furthermore, WNT10A overexpression renders cells resistant to apoptosis induced by hydrogen peroxide and high glucose. Collectively, WNT10A may induce kidney fibrosis and associate with kidney dysfunction in AIN.
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A taxonomic review of the Neoserica (sensu lato) septemlamellata group (Coleoptera, Scarabaeidae, Sericini).
Zookeys
PUBLISHED: 01-01-2014
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In the present paper the species belonging to the Neoserica (sensu lato) septemlamellata group, that included so far only four known species, are revised. Here we describe eleven new species originating mainly from Indochina and Southern China: N. daweishanica sp. n., N. gaoligongshanica sp. n., N. guangpingensis sp. n., N. igori sp. n., N. jiulongensis sp. n., N. plurilamellata sp. n., N. weishanica sp. n., N. yanzigouensis sp. n. (China) N. sapaensis sp. n. (China, Vietnam), N. bansongchana sp. n., N. takakuwai sp. n. (Laos). The lectotypes of Neoserica septemlamellata Brenske, 1898 and N. septemfoliata Moser, 1915 are designated. Keys to the species and species groups are given, the genitalia of all species and their habitus are illustrated and distribution maps are included.
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Association of saccade duration and saccade acceleration/deceleration asymmetry during visually guided saccade in schizophrenia patients.
PLoS ONE
PUBLISHED: 01-01-2014
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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.
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Template-Activated Strategy toward One-Step Coating Silica Colloidal Microspheres with Sliver.
ACS Appl Mater Interfaces
PUBLISHED: 12-27-2013
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Template-activated strategy was developed to coat silica (SiO2) colloidal microspheres with silver in one step, based on one-pot hydrothermal treatment of silver nitrate, PVP (poly(vinyl pyrrolidone)), and SiO2 colloidal microspheres in ammonia solution. In our reaction system, the surface of SiO2 colloidal microspheres was continually activated with negative-charged SiO(-) groups in ammonia solution, which accumulated [Ag(NH3)2](+) or Ag(+) ions around the SiO2 colloidal microspheres through electrostatic attraction; thereafter these ions could be reduced into Ag nanoparticles in situ by the weak reducer PVP in the solution, and then acted as seeds for the subsequent complete silver coating with the reaction proceeding. Therefore, the traditional three steps for complete silver coating, including prior surface modification, seeding, and subsequent growing, were effectively integrated into one step. The experimental results exhibited that perfect SiO2/Ag core/shell composite microspheres were successfully synthesized through optimizing the reaction parameters like the solvent ingredient, reducer, and the reaction temperature. Additionally, these obtained uniform composite microspheres were further used as SERS substrate by using R6G and thiram as probe molecules, and showed excellent trace detection of these organic chemicals in solution.
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Tumor-derived GM-CSF promotes inflammatory colon carcinogenesis via stimulating epithelial release of VEGF.
Cancer Res.
PUBLISHED: 12-23-2013
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Chronic inflammation is a major driving force for the development of colitis-associated cancer (CAC). Elevated production of granulocyte macrophage colony-stimulating factor (GMCSF) has been observed in mucosa of patients with inflammatory bowel disease (IBD). Its actions in the progression from colitis to cancer, however, remain poorly understood. Herein, we demonstrated that colonic epithelial cells (CEC) were a major cellular source of GMCSF and its production was significantly augmented when CAC model was established by administration of azoxymethane and dextran sulfate sodium. Furthermore, we showed that GMCSF was a driver for VEGF release by CEC in autocrine and/or paracrine manners through extracellular regulated protein kinase (ERK)-dependent pathway. Blocking GMCSF activity in vivo significantly decreased epithelial release of VEGF, thereby abrogated CAC formation. In vitro treatment of transformed CEC with recombinant GMCSF dramatically augmented its invasive potentials, largely in VEGF-dependent fashion. Furthermore, commensal microbiota-derived lipopolysaccharides (LPS) was identified a trigger for GMCSF expression in CEC, as antibiotics treatment or toll like receptor 4 (TLR4) ablation considerably impaired its epithelial expression. Overall, these findings may have important implications for understanding of mechanisms underlying CAC pathogenesis and the therapeutic potentials of regimens targeting GMCSF or VEGF in clinic.
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Down-regulation of Aquaporin1 (AQP1) by peptidoglycan via p38 MAPK pathways in primary rat pleural mesothelial cells.
Exp. Lung Res.
PUBLISHED: 12-23-2013
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ABSTRACT Background and objective. This study was designed to investigate the p38 mitogen-activated protein kinase (MAPK) signaling pathway involved in Aquaporin1 (AQP1) expression caused by staphylococcal peptidoglycan (PGN) in cultured rat pleural mesothelial cells (rPMCs) in vitro. Methods. RT-PCR and immunoblot analysis were used to determine the relative mRNA and protein levels of AQP1 by PGN in rPMCs. P38 kinase inhibitor SB203580, JNK inhibitor SP600125, and ERK1/2 inhibitor PD98059 were used to determine the effects of PGN-induced AQP1 expression by immunoblot. Activation of p38 by PGN was reflected by detecting the phosphorylation constituent of p38, using immunoblot. The shift of localization after activation of p38 by PGN was investigated by immunofluorescence assay. Results. AQP1 transcription and protein expression were decreased by PGN in dose-dependent and time-dependent manners in rPMCs. Down-regulation of AQP1 by PGN was blocked only by SB203580, neither by SP600125 nor by PD98059. Furthermore, rPMCs exposed to PGN showed activation of p38 MAPK. Phospho-p38 protein production was increased by PGN stimulation in rPMCs. The localization of phospho-p38 was both in the cytosol and nuclei after PGN treatment, while its normal distribution is mainly in the cytosol in rPMCs. Conclusion. AQP1 expression was decreased by PGN in both dose-dependent and time-dependent manners in rPMCs. This down-regulation by PGN-induced AQP1 in rPMCs may be mediated by the activation of p38 MARK pathway.
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Noninvasive Assessment of Left Atrial Phasic Function in Patients with Hypertension and Diabetes Using Two-Dimensional Speckle Tracking and Volumetric Parameters.
Echocardiography
PUBLISHED: 12-20-2013
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To evaluate the left atrial phasic function of hypertensive patients with or without coexisting diabetes using two-dimensional speckle tracking echocardiography (2DSTE)-based strain and strain rate imaging and volumetric parameters.
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In vivo three-photon microscopy of subcortical structures within an intact mouse brain.
Nat Photonics
PUBLISHED: 12-20-2013
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Two-photon fluorescence microscopy (2PM)(1) enables scientists in various fields including neuroscience(2,3), embryology(4), and oncology(5) to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue(3) or the insertion of optical probes(6,7). Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.
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Validation of a Venous Thromboembolism Risk Assessment Model in Hospitalized Chinese Patients: A Case-Control Study.
J. Atheroscler. Thromb.
PUBLISHED: 12-03-2013
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Aim: The appropriate selection of hospitalized patients for venous thromboembolism(VTE) prophylaxis is an important unresolved issue. We sought to validate the Caprini model, a famous individual VTE risk assessment model(RAM), in hospitalized Chinese patients. Methods: We performed a retrospective case-control study among unselected hospitalized patients admitted to a comprehensive hospital in China. A total of 347 patients were confirmed to have VTE during hospitalization, and 651 controls were randomly selected to match the patients according to medical service. Both the patients and controls were retrospectively assessed for the risk of VTE using the Caprini RAM. Results: The average Caprini cumulative risk score in the patients was significantly higher than that observed in the controls(4.69±2.58 vs 3.16±1.82, p?0.0001). Compared with that observed in the low-risk group, a classification of high-risk according to the Caprini model was associated with a 1.65-fold increased risk of VTE(95%CI 1.05-2.61), while that of highest-risk was associated with a 4.84-fold increased risk of VTE(95%CI 3.06-7.64). After further stratifying the highest risk level with a cumulative risk score of ?5 into scores of 5-6, 7-8 and ?9, the patients with a score of 5-6 were found to exhibit a 3.33-fold increased risk of VTE(95%CI 2.06-5.40), those with a score 7-8 exhibited a 9.41-fold increased risk of VTE(95%CI 4.90-18.08) and those with a score of ?9 exhibited a 24.69-fold(95%CI 7.98-76.40) increased risk of VTE compared with their low-risk counterparts. Conclusions: Our study suggests that the Caprini RAM can be used to effectively stratify hospitalized Chinese patients into VTE risk categories based on individual risk factors. The classification of the highest risk level with a cumulative risk score of ?5 provided significantly more clinical information, and further stratification of this group of patients is needed.
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