JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries.
J Vis Exp
PUBLISHED: 11-20-2014
Show Abstract
Hide Abstract
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.(1,2) However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.(3) This research is challenging, and we outline a method to address these challenges using in situ NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries. We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the 'roll-over' cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ NPD experiment and initial directions are presented on how to analyze such complex in situ data.
Related JoVE Video
Slope characterization in combining analog and photon count data from atmospheric lidar measurements.
Appl Opt
PUBLISHED: 11-18-2014
Show Abstract
Hide Abstract
A transient digitizer (Licel) connecting to a photomultiplier tube (PMT) can obtain lidar backscatter profiles in both photon count and analog signal. A lidar can detect higher atmospheric regions by combining (gluing) simultaneous analog and photon count data via the slope coefficient. In this work, the output of a PMT has been measured with a transient digitizer based on an intensity-stable light source. The slope value and dynamic range of the lidar profile have been characterized. The slope value is determined only by the gain of the PMT as it works in a linear range with a fixed pulse height discrimination threshold. The dynamic range of a glued lidar profile is settled by the slope value. The fitted slope has a more exact value when the selection criterion is given in terms of the independent variable for fitting. For practical lidar data, the fitted slope is more stable and reliable when the lower limit of the data range for fitting rises.
Related JoVE Video
Electrochemistry and structure of the cobalt-free Li1+xMO2 (M = Li, Ni, Mn, Fe) composite cathode.
Phys Chem Chem Phys
PUBLISHED: 10-22-2014
Show Abstract
Hide Abstract
The development of cathode materials with high capacity and cycle stability is essential to emerging electric-vehicle technologies, however, of serious environmental concern is that materials with these properties developed so far contain the toxic and expensive Co. We report here the Li-rich, Co-free Li1+xMO2 (M = Li, Ni, Mn, Fe) composite cathode material, prepared via a template-free, one-step wet-chemical method followed by conventional annealing in an oxygen atmosphere. The cathode has an unprecedented level of cation mixing, where the electrochemically-active component contains four elements at the transition-metal (3a) site and 20% Ni at the active Li site (3b). We find Ni(2+)/Ni(3+)/Ni(4+) to be the active redox-center of the cathode with lithiation/delithiation occurring via a solid-solution reaction where the lattice responds approximately linearly with cycling, differing to that observed for iso-structural commercial cathodes with a lower level of cation mixing. The composite cathode has ?75% active material and delivers an initial discharge-capacity of ?103 mA h g(-1) with a reasonable capacity retention of ?84.4% after 100 cycles. Notably, the electrochemically-active component possesses a capacity of ?139 mA h g(-1), approaching that of the commercialized LiCoO2 and Li(Ni1/3Mn1/3Co1/3)O2 materials. Importantly, our operando neutron powder-diffraction results suggest excellent structural stability of this active component, which exhibits ?80% less change in its stacking-axis than for LiCoO2 with approximately the same capacity, a characteristic that may be exploited to enhance significantly the capacity retention of this and similar materials.
Related JoVE Video
VMAT planning study in rectal cancer patients.
Radiat Oncol
PUBLISHED: 10-16-2014
Show Abstract
Hide Abstract
BackgroundTo compare the dosimetric differences among fixed field intensity-modulated radiation therapy (IMRT), single-arc volumetric-modulated arc therapy (SA-VMAT) and double-arc volumetric-modulated arc therapy (DA-VMAT) plans in rectal cancer.MethodFifteen patients with rectal cancer previously treated with IMRT in our institution were selected for this study. For each patient, three plans were generated with the planning CT scan: one using a fixed beam IMRT, and two plans using the VMAT technique: SA-VMAT and DA-VMAT. Dose prescription to the PTV was 50 Gy in 2 Gy per fraction. Dose volume histograms (DVH) for the target volume and the organs at risk (small bowel, bladder, femoral heads and healthy tissue) were compared for these different techniques. Monitor units (MU) and delivery treatment time were also reported.ResultsDA-VMAT achieved the highest minimum planning target volume (PTV) dose and the lowest maximal dose, resulting in the most homogeneous PTV dose distribution. DA-VMAT also yielded the best CI, although the difference was not statistically significant. Between SA-VMAT and IMRT, the target dose coverage was largely comparable; however, SA-VMAT was able to achieve a better V95 and V107. VMAT showed to be inferior to IMRT in terms of organ at risk sparing, especially for the small bowel. Compared with IMRT, DA-VMAT increased the V15 of small bowel nearly 55 cc. The MU and treatment delivery time were significantly reduced by the use of VMAT techniques.ConclusionVMAT is a new radiation technique that combines the ability to achieve highly conformal dose distributions with highly efficient treatment delivery. Considering the inferior role of normal tissue sparing, especially for small bowel, VMAT need further investigation in rectal cancer treatment.
Related JoVE Video
The CpxR/CpxA Two-Component Regulatory System Upregulates the Multidrug Resistance Cascade to Facilitate Escherichia coli Resistance to A Model Antimicrobial Peptide.
J. Biol. Chem.
PUBLISHED: 10-06-2014
Show Abstract
Hide Abstract
A genome-wide susceptibility assay was used to identify specific CpxR-dependent genes that facilitate Escherichia coli resistance to a model cationic antimicrobial peptide (CAMP), protamine. A total of 115 strains from the Keio Collection, each of which contained a deletion at a demonstrated or predicted CpxR/CpxA-dependent locus, were tested for protamine susceptibility. One strain that exhibited high susceptibility carried a deletion of tolC, a gene that encodes the outer membrane component of multiple tripartite multidrug transporters. Concomitantly, two of these efflux systems, AcrAB/TolC and EmrAB/TolC, play major roles in protamine resistance. Activation of the CpxR/CpxA system stimulates mar transcription, suggesting a new regulatory circuit that enhances the multidrug resistance cascade. Tripartite multidrug efflux systems contribute to bacterial resistance to protamine differently from the Tat system. DNase I footprinting analysis demonstrated that the CpxR protein binds to a sequence located in the -35 and -10 regions of mar promoter. This sequence resembles the consensus CpxR binding site, however, on the opposite strand. aroK, a CpxR-dependent gene that encodes a shikimate kinase in the tryptophan biosynthesis pathway, was also found to facilitate protamine resistance. Specific aromatic metabolites from this pathway, such as indole, can stimulate expression of well-studied CpxR-dependent genes degP and cpxP, which are not components of the tripartite multidrug transporters. Thus, we propose a novel mechanism for E. coli to modulate resistance to protamine and likely other CAMPs, in which the CpxR/CpxA system upregulates mar transcription in response to specific aromatic metabolites, subsequently stimulating the multidrug resistance cascade.
Related JoVE Video
Effects and mechanism of suberoylanilide hydroxamic acid on the proliferation and apoptosis of human hepatoma cell line Bel-7402.
J BUON
PUBLISHED: 09-28-2014
Show Abstract
Hide Abstract
To investigate the effects and mechanism of suberoylanilide hydroxamic acid (SAHA) on the proliferation and apoptosis of human hepatoma cell line Bel-7402.
Related JoVE Video
[Significant genes extraction and analysis of gene expression data based on matrix factorization techniques].
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
PUBLISHED: 09-16-2014
Show Abstract
Hide Abstract
It is generally considered that various regulatory activities between genes are contained in the gene expression datasets. Therefore, the underlying gene regulatory relationship and the biologically useful information can be found by modeling the gene regulatory network from the gene expression data. In our study, two unsupervised matrix factorization methods, independent component analysis (ICA) and nonnegative matrix factorization (NMF), were proposed to identify significant genes and model the regulatory network using the microarray gene expression data of Alzheimer's disease (AD). By bio-molecular analyzing of the pathways, the differences between ICA and NMF have been explored and the fact, which the inflammatory reaction is one of the main pathological mechanisms of AD, is also emphasized. It was demonstrated that our study gave a novel and valuable method for the research of early detection and pathological mechanism, biomarkers' findings of AD.
Related JoVE Video
Berberine Up-Regulates Hepatic Low-Density Lipoprotein Receptor through Ras-Independent but AMP-Activated Protein Kinase-Dependent Raf-1 Activation.
Biol. Pharm. Bull.
PUBLISHED: 09-05-2014
Show Abstract
Hide Abstract
Our previous studies showed that berberine (BBR) increases liver low-density lipoprotein (LDL) receptor expression in an extracellular signal-regulated kinase (ERK)-dependent manner. This study was designed to explore the upstream cellular signaling molecules recruited by BBR to activate the ERK mitogen-activated protein kinase (MAPK) cascade. Chemical inhibitors such as GW5074, manumycin A, and compound C or specific small interfering RNAs (siRNAs) were used in the blocking experiments; Western blot was used to determine the phosphorylation of kinases; real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression level of LDL receptor mRNA. Our results indicate that BBR increases p-Raf-1 (ser338) level time and dose dependently in HL-7702 cells, but has no influence on Ras activity; the stimulating activities of BBR on Raf-1 signaling and LDL receptor expression can be blocked by GW5074 completely, but not by manumycin A, a Ras inhibitor. BBR activates hepatic Raf-1 signaling and up-regulates LDL receptor expression in a rat model of hyperlipidemia with no impact on liver Ras activity. Importantly, our results show that the stimulating activities of BBR on hepatic Raf-1 signaling and LDL receptor expression are totally blocked by compound C, a selective inhibitor of AMP-activated protein kinase (AMPK), and also by silencing its expression with siRNA. Taken together, our results demonstrate for the first time that BBR up-regulates LDL receptor expression through Ras-independent, but AMPK-dependent Raf-1 activation in liver cells. Our study will help to elucidate the molecular pharmacology of BBR and provide new scientific evidence for its clinical application.
Related JoVE Video
Effects of IL-17 on expression of GRO-? and IL-8 in fibroblasts from nasal polyps.
J. Huazhong Univ. Sci. Technol. Med. Sci.
PUBLISHED: 08-19-2014
Show Abstract
Hide Abstract
Recent studies indicated that interleukin (IL)-17, growth-related oncogene (GRO)-? and IL-8 play an important role in the pathogenesis of nasal polyps. However, the effects of the increased amount of IL-17 and the production of GRO-? and IL-8 in human nasal polyp fibroblasts are not completely understood. This study aimed to determine the effects of the increased IL-17 on the changes of GRO-? and IL-8 expression in human nasal polyp fibroblasts and further investigate the mechanism of neutrophil infiltration in nasal polyps. Nasal polyp fibroblasts were isolated from six cases of human nasal polyps, and the cells were stimulated with five different concentrations of IL-17. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of GRO-? and IL-8. The mRNA of GRO-? and IL-8 was expressed in unstimulated controls and remarkably increased by stimulation with IL-17. Moreover, the levels of GRO-? and IL-8 produced by fibroblasts were increased gradually with the increases in IL-17 concentrations. The present study showed that nasal fibroblasts can produce GRO-? and IL-8, and their production is remarkably enhanced by IL-17 stimulation, thereby clarifying the mechanism of the IL-17 mediated neutrophil infiltration in nasal polyps. These findings might provide a rationale for using IL-17 inhibitors as a treatment for nasal inflammatory diseases such as nasal polyps.
Related JoVE Video
Age-dependent expression of duodenal cytochrome B561, divalent metal transporter 1, ferroportin 1 and hephaestin in the duodenum of rats.
J. Gastroenterol. Hepatol.
PUBLISHED: 08-12-2014
Show Abstract
Hide Abstract
The body's requirement for iron is different at different developmental stages. However, the molecular mechanisms of age-dependent iron metabolism are poorly understood. In the present study, we investigated the expression of iron transport proteins in the duodenum of Sprague-Dawley (SD) rats at five different age stages.
Related JoVE Video
A bioinformatics method for predicting long noncoding RNAs associated with vascular disease.
Sci China Life Sci
PUBLISHED: 08-08-2014
Show Abstract
Hide Abstract
Long noncoding RNAs (lncRNAs) play important roles in human diseases including vascular disease. Given the large number of lncRNAs, however, whether the majority of them are associated with vascular disease remains unknown. For this purpose, here we present a genomic location based bioinformatics method to predict the lncRNAs associated with vascular disease. We applied the presented method to globally screen the human lncRNAs potentially involved in vascular disease. As a result, we predicted 3043 putative vascular disease associated lncRNAs. To test the accuracy of the method, we selected 10 lncRNAs predicted to be implicated in proliferation and migration of vascular smooth muscle cells (VSMCs) for further experimental validation. The results confirmed that eight of the 10 lncRNAs (80%) are validated. This result suggests that the presented method has a reliable prediction performance. Finally, the presented bioinformatics method and the predicted vascular disease associated lncRNAs together may provide helps for not only better understanding of the roles of lncRNAs in vascular disease but also the identification of novel molecules for the diagnosis and therapy of vascular disease.
Related JoVE Video
Detoxified pneumolysin derivative Plym2 directly protects against pneumococcal infection via induction of inflammatory cytokines.
Immunol. Invest.
PUBLISHED: 07-14-2014
Show Abstract
Hide Abstract
Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.
Related JoVE Video
Enhanced sodium-ion battery performance by structural phase transition from two-dimensional hexagonal-SnS2 to orthorhombic-SnS.
ACS Nano
PUBLISHED: 07-14-2014
Show Abstract
Hide Abstract
Structural phase transitions can be used to alter the properties of a material without adding any additional elements and are therefore of significant technological value. It was found that the hexagonal-SnS2 phase can be transformed into the orthorhombic-SnS phase after an annealing step in an argon atmosphere, and the thus transformed SnS shows enhanced sodium-ion storage performance over that of the SnS2, which is attributed to its structural advantages. Here, we provide the first report on a SnS@graphene architecture for application as a sodium-ion battery anode, which is built from two-dimensional SnS and graphene nanosheets as complementary building blocks. The as-prepared SnS@graphene hybrid nanostructured composite delivers an excellent specific capacity of 940 mAh g(-1)and impressive rate capability of 492 and 308 mAh g(-1) after 250 cycles at the current densities of 810 and 7290 mA g(-1), respectively. The performance was found to be much better than those of most reported anode materials for Na-ion batteries. On the basis of combined ex situ Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and ex situ X-ray diffraction, the formation mechanism of SnS@graphene and the synergistic Na-storage reactions of SnS in the anode are discussed in detail. The SnS experienced a two-structural-phase transformation mechanism (orthorhombic-SnS to cubic-Sn to orthorhombic-Na3.75Sn), while the SnS2 experienced a three-structural-phase transformation mechanism (hexagonal-SnS2 to tetragonal-Sn to orthorhombic-Na3.75Sn) during the sodiation process. The lesser structural changes of SnS during the conversion are expected to lead to good structural stability and excellent cycling stability in its sodium-ion battery performance. These results demonstrate that the SnS@graphene architecture offers unique characteristics suitable for high-performance energy storage application.
Related JoVE Video
Zero kinetic energy photoelectron spectroscopy of triphenylene.
J Chem Phys
PUBLISHED: 07-03-2014
Show Abstract
Hide Abstract
We report vibrational information of both the first electronically excited state and the ground cationic state of jet-cooled triphenylene via the techniques of resonantly enhanced multiphoton ionization (REMPI) and zero kinetic energy (ZEKE) photoelectron spectroscopy. The first excited electronic state S1 of the neutral molecule is of A1' symmetry and is therefore electric dipole forbidden in the D3h group. Consequently, there are no observable Franck-Condon allowed totally symmetric a1' vibrational bands in the REMPI spectrum. All observed vibrational transitions are due to Herzberg-Teller vibronic coupling to the E' third electronically excited state S3. The assignment of all vibrational bands as e' symmetry is based on comparisons with calculations using the time dependent density functional theory and spectroscopic simulations. When an electron is eliminated, the molecular frame undergoes Jahn-Teller distortion, lowering the point group to C2v and resulting in two nearly degenerate electronic states of A2 and B1 symmetry. Here we follow a crude treatment by assuming that all e' vibrational modes resolve into b2 and a1 modes in the C2v molecular frame. Some observed ZEKE transitions are tentatively assigned, and the adiabatic ionization threshold is determined to be 63?365 ± 7 cm(-1). The observed ZEKE spectra contain a consistent pattern, with a cluster of transitions centered near the same vibrational level of the cation as that of the intermediate state, roughly consistent with the propensity rule. However, complete assignment of the detailed vibrational structure due to Jahn-Teller coupling requires much more extensive calculations, which will be performed in the future.
Related JoVE Video
[Determination of ochratoxin A in human urine by HPLC-FLD after cleaned-up by molecularly imprinted polymer solid phase extraction column].
Yao Xue Xue Bao
PUBLISHED: 07-01-2014
Show Abstract
Hide Abstract
A method was developed for the determination of ochratoxin A (OTA) in human urine by HPLC-FLD after molecularly imprinted polymer solid phase extraction (MIP-SPE) column. After the pH being adjusted to 2.5 with 0.1 mol x L(-1) HC1, sample was cleaned up with MIP-SPE column for ochratoxin A, the analyte was analyzed by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD), and finally all the positive results were confirmed by LC-MS/MS. Recoveries from urine samples spiked with OTA at levels ranging from 2 to 20 ng x mL(-1) were 90.6%-101.9%, and RSDs were 0.1%-1.6%. Sixty-five volunteers living in Beijing took part in the study, of which 5 were found containing OTA in their urine and the highest value was 0.091 ng x mL(-1). The MIP-SPE column was firstly applied to purify and concentrate OTA in human urine, this method is simple, rapid and reliable and can be used to determine the contents of OTA in human urine.
Related JoVE Video
Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.
Retrovirology
PUBLISHED: 06-19-2014
Show Abstract
Hide Abstract
BackgroundFitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.ResultsThe T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.ConclusionsOur results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.
Related JoVE Video
Adjuvant chemoradiotherapy versus chemotherapy for gastric cancer: A meta-analysis of randomized controlled trials.
J Surg Oncol
PUBLISHED: 06-12-2014
Show Abstract
Hide Abstract
The aim of this study was to evaluate the efficacy and safety of adjuvant chemoradiotherapy (CRT) versus chemotherapy (CT) for patients with gastric cancer.
Related JoVE Video
Pd-catalyzed regio- and stereoselective addition of boronic acids to silylacetylenes: a stereodivergent assembly of ?,?-disubstituted alkenylsilanes and alkenyl halides.
J. Org. Chem.
PUBLISHED: 06-11-2014
Show Abstract
Hide Abstract
An efficient Pd-catalyzed addition of boronic acids to silylacetylenes is described, providing ?,?-disubstituted (E)- or (Z)-alkenylsilanes in satisfactory yields with excellent regio- and stereoselectivity under mild reaction conditions. It represents the first highly regio- and stereoselective addition of boronic acids to aryl and alkenyl silylacetylenes. Moreover, the sequential Pd-catalyzed boron addition/N-halosuccinimide-mediated halodesilylation reaction results in a stereodivergent approach to ?,?-disubstituted alkenyl halides, which can serve as versatile synthetic intermediates for the stereodivergent assembly of (E)- and (Z)-trisubstituted alkenes via transition-metal-catalyzed cross-coupling reactions.
Related JoVE Video
Electrospun P2-type Na(2/3)(Fe(1/2)Mn(1/2))O2 hierarchical nanofibers as cathode material for sodium-ion batteries.
ACS Appl Mater Interfaces
PUBLISHED: 06-10-2014
Show Abstract
Hide Abstract
Sodium-ion batteries can be the best alternative to lithium-ion batteries, because of their similar electrochemistry, nontoxicity, and elemental abundance and the low cost of sodium. They still stand in need of better cathodes in terms of their structural and electrochemical aspects. Accordingly, the present study reports the first example of the preparation of Na2/3(Fe1/2Mn1/2)O2 hierarchical nanofibers by electrospinning. The nanofibers with aggregated nanocrystallites along the fiber direction have been characterized structurally and electrochemically, resulting in enhanced cyclability when compared to nanoparticles, with initial discharge capacity of ?195 mAh g(-1). This is attributed to the good interconnection among the fibers, with well-guided charge transfers and better electrolyte contacts.
Related JoVE Video
Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling.
J. Am. Soc. Mass Spectrom.
PUBLISHED: 05-19-2014
Show Abstract
Hide Abstract
An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway.
Related JoVE Video
Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.
Viruses
PUBLISHED: 05-13-2014
Show Abstract
Hide Abstract
Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability?based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 ?M. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.
Related JoVE Video
Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins.
Retrovirology
PUBLISHED: 05-03-2014
Show Abstract
Hide Abstract
BackgroundAll lentiviruses except equine infectious anemia virus (EIVA) antagonize antiviral family APOBEC3 (A3) proteins of the host through viral Vif proteins. The mechanism by which Vif of human, simian or feline immunodeficiency viruses (HIV/SIV/FIV) suppresses the corresponding host A3s has been studied extensively.ResultsHere, we determined that bovine immunodeficiency virus (BIV) and maedi-visna virus (MVV) Vif proteins utilize the Cullin (Cul)-ElonginB (EloB)-ElonginC (EloC) complex (BIV Vif recruits Cul2, while MVV Vif recruits Cul5) to degrade Bos taurus (bt)A3Z2-Z3 and Ovis aries (oa)A3Z2-Z3, respectively, via a proteasome-dependent but a CBF-ß-independent pathway. Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC and oaEloC and dominant-negative mutants of btCul2 and oaCul5 could disrupt the activity of BIV and MVV Vif, respectively. While the membrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation of btA3Z2-Z3, it had minimal effects on oaA3Z2-Z3 degradation induced by MVV Vif, indicating that Zn is important for the activity of BIV Vif but not MVV Vif. Furthermore, we identified a previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC box which is critical for its degradation activity.ConclusionsA novel zinc binding loop was identified in the BIV Vif protein that is important for the E3 ubiquination activity, suggesting that the degradation of btA3Z2-Z3 by BIV and that of oaA3Z2-Z3 by MVV Vif may need host factors other than CBF-ß.
Related JoVE Video
Permeability of exendin-4-loaded chitosan nanoparticles across MDCK cell monolayers and rat small intestine.
Biol. Pharm. Bull.
PUBLISHED: 05-03-2014
Show Abstract
Hide Abstract
The purpose of this study was to investigate the permeability of exendin-4-loaded chitosan nanoparticles using the Madin Darby canine kidney (MDCK) cell monolayer as an in vitro model and the rat intestine as an ex vivo model of the human intestinal barrier. A series of formulations of sodium tripolyphosphate (TPP) and chitosan with different molecular weights and degrees of deacetylation was evaluated. The formulation consisting of 0.1% TPP and 0.2% chitosan (400?kDa, 95% degree of deacetylation), which gave optimized monodispersed particle size (303.1±10.36?nm), zeta potential (18.37±1.15?mV) and encapsulation efficiency (38.0±2.6%), was used for further analysis. After determining their biocompatibility, the transport potential of drug-loaded chitosan nanoparticles was evaluated and compared with free exendin-4 using both MDCK cell monolayers and different rat intestinal segments. Mechanisms underlying enhanced transport of exendin-4 in the cell model were also explored. Compared with free exendin-4, the absorption of optimized chitosan nanoparticles was enhanced by 4.7-fold in MDCK cell monolayers and by 2.0-2.78-fold in different rat intestinal segments, with no significant difference between the duodenum, jejunum and ileum. As supported by confocal laser scanning microscopic analysis, the lower enhancement of absorption in the intestine compared to the cell monolayer likely resulted from the chitosan nanoparticle-mediated opening of cellular tight junctions and not through intracellular transport. These findings suggest that the potential application of chitosan nanoparticles as delivery carriers of exendin-4 is limited and may need further modifications.
Related JoVE Video
The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 05-02-2014
Show Abstract
Hide Abstract
Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai(2+) transients measured using high-affinity Ca(2+)-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai(2+) measurements in intact adult cardiac tissue by comparing optical recordings obtained with high- and low-affinity dyes. Experiments were carried out in porcine left ventricular (LV) wedge preparations stained locally by intramural injection via microcapillaries (diameter = 150 ?m) with a low-affinity Ca(2+)-sensitive dye Fluo-4FF or Fluo-2LA (nominal Kd, ~7-10 ?mol/l), high-affinity dye Rhod-2 (Kd = 0.57 ?mol/l), and Fluo-4 or Fluo-2MA (Kd, ~0.4 ?mol/l); in addition, tissue was stained with transmembrane potential (Vm)-sensitive dye RH-237. Optical recordings of Vm and Cai(2+) were made using optical fibers (diameter = 325 ?m) glued with the microcapillaries. The durations of Cai(2+) transients measured at 50% level of recovery (CaD50) using high-affinity Fluo-4/Fluo-2MA dyes were up to ~81% longer than those measured with low-affinity Fluo-4FF/Fluo-2LA at long pacing cycle lengths (CL). In Fluo-4/Fluo-2MA measurements at long CLs, Cai(2+) transients often (~50% of cases) exhibited slow upstroke rise and extended plateau. In Rhod-2 measurements, CaD50 was moderately longer (up to ~35%) than in Fluo-4FF recordings, but Cai(2+) transient shapes were similar. In all series of measurements, mean action potential duration values were not significantly different (P > 0.05). The delays between Vm and Cai(2+) upstrokes were comparable for low- and high-affinity dyes (P > 0.05). In conclusion, measurements of Cai(2+) transient in ventricular myocardium are strongly affected by the affinity of Ca(2+) dyes. The high-affinity dyes may overestimate the duration and alter the shape of Cai(2+) transients.
Related JoVE Video
Trimeric knob protein specifically distinguishes neutralizing antibodies to different human adenovirus species: potential application for adenovirus seroepidemiology.
J. Gen. Virol.
PUBLISHED: 04-24-2014
Show Abstract
Hide Abstract
Adenoviruses (Ads) are non-enveloped DNA viruses that have been extensively studied and used as vectors for gene therapy and several potential vaccines. There are 57 Ad serotypes in seven species (A-G), and Ad neutralizing antibody (NAb) titres can vary by serotype and geographical location. Until now serotype- and species-specific antibodies have been detected by neutralization or haemagglutination inhibition assays. These expensive and cumbersome methods of adenovirus typing have mainly been used in epidemiological studies. Our prior work demonstrated that NAbs against the fiber protein are commonly generated during natural Ad infection in humans and the trimeric knob is preferentially recognized by fiber-induced NAbs. In this study, we expressed nine trimeric knob proteins from representative Ad serotypes of human Ad (HAdV)-A-F in Escherichia coli and found no cross-reactivity of these recombinant proteins with rabbit hyperimmune sera (among HAdV-A-F or within HAdV-C). Results of the ELISA based on Ad2 and Ad5 (both HAdV-C) knob proteins were consistent with those of neutralization assays, indicating that the trimeric knob protein would be a good candidate antigen for detecting Ad serotype-specific NAbs in sera from naturally infected subjects. We also demonstrated the primary seroepidemiology of nine Ad serotypes in 274 children using the knob-based ELISA. These results have potential implications for epidemiology of Ad serotypes and future development of Ad-based vaccines and gene therapy.
Related JoVE Video
NLRP3 inflammasome sequential changes in Staphylococcus aureus-induced mouse model of acute rhinosinusitis.
Int J Mol Sci
PUBLISHED: 04-17-2014
Show Abstract
Hide Abstract
The NLR pyrin domain containing 3 (NLRP3) inflammasome plays a crucial role in lung disease and may have a similar role in upper respiratory tract inflammation. We therefore constructed a C57BL/6 mouse model of acute rhinosinusitis induced by Staphylococcus aureus and investigated the role of the NLRP3 inflammasome in this model. Mice were classified as non-inoculated group (group A) and inoculated groups (groups B, C, D and E, sacrificed 1, 3, 7 and 14 days after inoculation, respectively). Hematoxylin-eosin staining showed that each group had inflammatory cell infiltration, except group A. The damage of the nasal mucosa was aggravated gradually over time. Western blot and immunofluorescence showed that the structural proteins of the NLRP3 inflammasome (NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), procaspase-1) in groups B, C, D and E were increased gradually. But they were reduced in group B compared with group A, except for NLRP3. Western blot showed that the cleavage fragment of procaspase-1, p20 in groups B, C, D and E was increased gradually. Real-time PCR showed that the corresponding mRNAs of the structural proteins were changed the same as their proteins. IL-1? mRNA and mature IL-1? protein were increased gradually in groups A, B, C, D and E. These results indicate that NLRP3 inflammasome activation was associated with the acute rhinosinusitis, and that there was a positive correlation between the expression level of the NLRP3 inflammasome and the severity of acute rhinosinusitis.
Related JoVE Video
Electron Diffraction of Superfluid Helium Droplets.
J Phys Chem Lett
PUBLISHED: 04-07-2014
Show Abstract
Hide Abstract
We present experimental results of electron diffraction of superfluid helium droplets and droplets doped with phthalocyanine gallium chloride and discuss the possibility of performing the same experiment with a laser aligned sample. The diffraction profile of pure droplets demonstrates dependence on the nozzle temperature, that is, on the average size of the droplets. Larger clusters demonstrate faster decay with increasing momentum transfer, whereas smaller clusters converge to isolated gas phase molecules at source temperatures of 18 K and higher. Electron diffraction of doped droplets shows similar modified molecular scattering intensity as that of the corresponding gas phase molecules. On the basis of fittings of the scattering profile, the number of remaining helium atoms of the doped droplets is estimated to be on the order of hundreds. This result offers guidance in assessing the possibility of electron diffraction from laser aligned molecules doped in superfluid helium droplets.
Related JoVE Video
The prognostic value of ERCC1 expression in gastric cancer patients treated with platinum-based chemotherapy: a meta-analysis.
Tumour Biol.
PUBLISHED: 03-16-2014
Show Abstract
Hide Abstract
Numerous studies examined the association between excision repair complementation group 1 (ERCC1) expression and the prognosis of gastric cancer patients receiving platinum-based chemotherapy but yielded controversial results. We thus conducted a meta-analysis to quantitatively evaluate the prognostic value of ERCC1 expression in gastric cancer patients receiving platinum-based chemotherapy. A systematic literature search was performed to identify relevant studies in PubMed, Web of Science, Embase, Cochrane Library, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, and WanFang Database up to December 17, 2013. Pooled hazard ratios (HRs) or odds ratios (ORs) with 95 % confidence intervals (CIs) were estimated. Moreover, meta-regression analysis and subgroup analysis were conducted according to ethnicity, HR extraction, detection methods, survival analysis, and quality score. A total of 1,409 patients from 21 studies were subjected to final analysis. Positive/high ERCC1 expression was significantly associated with poorer overall survival (HR, 1.58; 95 % CI, 1.09-2.28), especially in Asians (HR, 1.81; 95 % CI, 1.20-2.73), and lower response rate (OR, 0.26; 95 % CI, 0.18-0.36), but not with clinicopathological features, such as gender (OR, 1.01; 95 % CI, 0.68-1.51), grade (OR, 0.66; 95 % CI, 0.43-1.01), and stage (OR, 1.05; 95 % CI, 0.58-1.90). This meta-analysis suggested that ERCC1 expression might be a useful biomarker to predict response and survival for gastric cancer patients receiving platinum-based chemotherapy, particularly in Asians.
Related JoVE Video
Genetic analysis of riboswitch-mediated transcriptional regulation responding to Mn2+ in Salmonella.
J. Biol. Chem.
PUBLISHED: 03-04-2014
Show Abstract
Hide Abstract
Riboswitches are a class of cis-acting regulatory RNAs normally characterized from the 5'-UTR of bacterial transcripts that bind a specific ligand to regulate expression of associated genes by forming alternative conformations. Here, we present a riboswitch that contributes to transcriptional regulation through sensing Mn(2+) in Salmonella typhimurium. We characterized a 5'-UTR (UTR1) from the mntH locus encoding a Mn(2+) transporter, which forms a Rho-independent terminator to implement transcription termination with a high Mn(2+) selectivity both in vivo and in vitro. Nucleotide substitutions that cause disruption of the terminator interfere with the regulatory function of UTR1. RNA probing analyses outlined a specific UTR1 conformation that favors the terminator structure in Mn(2+)-replete condition. Switch sequence GCUAUG can alternatively base pair duplicated hexanucleotide CAUAGC to form either a pseudoknot or terminator stem. Mn(2+), but not Mg(2+), and Ca(2+), can enhance cleavage at specific nucleotides in UTR1. We conclude that UTR1 is a riboswitch that senses cytoplasmic Mn(2+) and therefore participates in Mn(2+)-responsive mntH regulation in Salmonella. This riboswitch domain is also conserved in several Gram-negative enteric bacteria, indicating that this Mn(2+)-responsive mechanism could have broader implications in bacterial gene expression. Additionally, a high level of cytoplasmic Mn(2+) can down-regulate transcription of the Salmonella Mg(2+) transporter mgtA locus in a Mg(2+) riboswitch-dependent manner. On the other hand, these two types of cation riboswitches do not share similarity at the primary or secondary structural levels. Taken together, characterization of Mn(2+)-responsive riboswitches should expand the scope of RNA regulatory elements in response to inorganic ions.
Related JoVE Video
Integrating gene expression and protein interaction data for signaling pathway prediction of Alzheimer's disease.
Comput Math Methods Med
PUBLISHED: 02-23-2014
Show Abstract
Hide Abstract
Discovering the signaling pathway and regulatory network would provide significant advance in genome-wide understanding of pathogenesis of human diseases. Despite the rich transcriptome data, the limitation for microarray data is unable to detect changes beyond transcriptional level and insufficient in reconstructing pathways and regulatory networks. In our study, protein-protein interaction (PPI) data is introduced to add molecular biological information for predicting signaling pathway of Alzheimer's disease (AD). Combining PPI with gene expression data, significant genes are selected by modified linear regression model firstly. Then, according to the biological researches that inflammation reaction plays an important role in the generation and deterioration of AD, NF- ? B (nuclear factor-kappa B), as a significant inflammatory factor, has been selected as the beginning gene of the predicting signaling pathway. Based on that, integer linear programming (ILP) model is proposed to reconstruct the signaling pathway between NF- ? B and AD virulence gene APP (amyloid precursor protein). The results identify 6 AD virulence genes included in the predicted inflammatory signaling pathway, and a large amount of molecular biological analysis shows the great understanding of the underlying biological process of AD.
Related JoVE Video
Overcoming the limitations of directed C-H functionalizations of heterocycles.
Nature
PUBLISHED: 02-18-2014
Show Abstract
Hide Abstract
In directed C-H activation reactions, any nitrogen or sulphur atoms present in heterocyclic substrates will coordinate strongly with metal catalysts. This coordination, which can lead to catalyst poisoning or C-H functionalization at an undesired position, limits the application of C-H activation reactions in heterocycle-based drug discovery, in which regard they have attracted much interest from pharmaceutical companies. Here we report a robust and synthetically useful method that overcomes the complications associated with performing C-H functionalization reactions on heterocycles. Our approach employs a simple N-methoxy amide group, which serves as both a directing group and an anionic ligand that promotes the in situ generation of the reactive PdX2 (X = ArCONOMe) species from a Pd(0) source using air as the sole oxidant. In this way, the PdX2 species is localized near the target C-H bond, avoiding interference from any nitrogen or sulphur atoms present in the heterocyclic substrates. This reaction overrides the conventional positional selectivity patterns observed with substrates containing strongly coordinating heteroatoms, including nitrogen, sulphur and phosphorus. Thus, this operationally simple aerobic reaction demonstrates that it is possible to bypass a fundamental limitation that has long plagued applications of directed C-H activation in medicinal chemistry.
Related JoVE Video
Hyperhomocysteinemia and methylenetetrahydrofolate reductase polymorphism in cervical artery dissection: a meta-analysis.
Cerebrovasc. Dis.
PUBLISHED: 02-18-2014
Show Abstract
Hide Abstract
Cervical artery dissection (CAD) is a recognized cause of ischemic stroke. Hyperhomocysteinemia (HHcy), i.e. an elevated concentration of plasma homocysteine, is identified as an independent risk factor for stroke prevalence. However, an association between HHcy and CAD has so far remained unknown.
Related JoVE Video
Synthesis of (Z)-1-thio- and (Z)-2-thio-1-alkenyl boronates via copper-catalyzed regiodivergent hydroboration of thioacetylenes: an experimental and theoretical study.
J. Org. Chem.
PUBLISHED: 02-10-2014
Show Abstract
Hide Abstract
A Cu-catalyzed divergent hydroboration of thioacetylenes has been achieved, providing (Z)-1-thio- or (Z)-2-thio-1-alkenyl boronates in moderate to high yields with excellent regio- and stereoselectivity, by using pinacolborane or bis(pinacolato)diboron as the hydroborating reagents, respectively. DFT calculations indicate that the sulfur atom plays a key role in determining the regioselectivity through polarizing the C-C triple bonds and participating in the HOMO orbitals. Moreover, the SR group can serve as a good leaving group, resulting in the concise synthesis of six regio- and stereoisomers of trisubstituted alkenes 5 via the iterative cross-coupling of C-B and C-S bonds. Clearly, it will be valuable for assembling stereochemically diverse trisubstituted olefins in organic synthesis.
Related JoVE Video
Development of a norovirus p particle platform for eliciting neutralizing antibody responses to the membrane proximal external region of human immunodeficiency virus type 1 envelope.
Protein Pept. Lett.
PUBLISHED: 02-09-2014
Show Abstract
Hide Abstract
Eliciting efficient broadly neutralizing antibodies (BnAbs) is an important goal that has yet to be achieved for human immunodeficiency type 1 (HIV-1) vaccine development, although they are rarely produced in virus-infected individuals. In particular, inducing specific neutralizing antibodies to the gp41 membrane proximal external region (MPER) has proven a difficult task. In this study, we introduce Norovirus P particles as a new platform to display the MPER epitope of HIV-1 as a vaccine with the aim of enhancing immune responses. The results showed that HIV-1 chimeric P particles were capable of inducing MPER-specific antibody responses in immunized guinea pigs, although only weakly neutralizing activity could be detected. These findings are consistent with other previous studies which have also focused on the well-studied 2F5 and 4E10 BnAbs. Our findings provide an alternate strategy for design of vaccines against HIV-1. However, great challenges remain in the effort to develop vaccines that can induce efficient HIV-1 neutralizing antibodies.
Related JoVE Video
Sex differences in iron status and hepcidin expression in rats.
Biol Trace Elem Res
PUBLISHED: 02-02-2014
Show Abstract
Hide Abstract
Studies have shown that men and women exhibit significant differences regarding iron status. However, the effects of sex on iron accumulation and distribution are not well established. In this study, female and male Sprague-Dawley rats were killed at 4 months of age. Blood samples were analyzed to determine the red blood cell (RBC) count, hemoglobin (Hb) concentration, hematocrit (Hct), and mean red blood cell volume (MCV). The serum samples were analyzed to determine the concentrations of serum iron (SI), transferrin saturation (TS), ferritin, soluble transferrin receptor (sTfR), and erythropoietin (EPO). The tissue nonheme iron concentrations were measured in the liver, spleen, bone marrow, kidney, heart, gastrocnemius, duodenal epithelium, lung, pallium, cerebellum, hippocampus, and striatum. Hepatic hepcidin expression was detected by real-time PCR analysis. The synthesis of ferroportin 1 (FPN1) in the liver, spleen, kidney, and bone marrow was determined by Western blot analysis. The synthesis of duodenal cytochrome B561 (DcytB), divalent metal transporter 1 (DMT1), FPN1, hephaestin (HP) in the duodenal epithelium was also measured by Western blot analysis. The results showed that the RBC, Hb, and Hct in male rats were higher than those in female rats. The SI and plasma TS levels were lower in male rats than in female rats. The levels of serum ferritin and sTfR were higher in male rats than in female rats. The EPO levels in male rats were lower than that in female rats. The nonheme iron contents in the liver, spleen, bone marrow, and kidney in male rats were also lower (56.7, 73.2, 60.6, and 61.4 % of female rats, respectively). Nonheme iron concentrations in the heart, gastrocnemius, duodenal epithelium, lung, and brain were similar in rats of both sexes. A moderate decrease in hepatic hepcidin mRNA content was also observed in male rats (to 56.0 % of female rats). The levels of FPN1 protein in the liver, spleen, and kidney were higher in male rats than in female rats. There was no significant change in FPN1 expression in bone marrow. Significant difference was also not found in DcytB, DMT1, FPN1, and HP protein levels in the duodenal epithelium between male and female rats. These data suggest that iron is distributed differently in male and female rats. This difference in iron distribution may be associated with the difference in the hepcidin level.
Related JoVE Video
Dynamic regulatory network reconstruction for Alzheimer's disease based on matrix decomposition techniques.
Comput Math Methods Med
PUBLISHED: 01-18-2014
Show Abstract
Hide Abstract
Alzheimer's disease (AD) is the most common form of dementia and leads to irreversible neurodegenerative damage of the brain. Finding the dynamic responses of genes, signaling proteins, transcription factor (TF) activities, and regulatory networks of the progressively deteriorative progress of AD would represent a significant advance in discovering the pathogenesis of AD. However, the high throughput technologies of measuring TF activities are not yet available on a genome-wide scale. In this study, based on DNA microarray gene expression data and a priori information of TFs, network component analysis (NCA) algorithm is applied to determining the TF activities and regulatory influences on TGs of incipient, moderate, and severe AD. Based on that, the dynamical gene regulatory networks of the deteriorative courses of AD were reconstructed. To select significant genes which are differentially expressed in different courses of AD, independent component analysis (ICA), which is better than the traditional clustering methods and can successfully group one gene in different meaningful biological processes, was used. The molecular biological analysis showed that the changes of TF activities and interactions of signaling proteins in mitosis, cell cycle, immune response, and inflammation play an important role in the deterioration of AD.
Related JoVE Video
Ag(I)-bovine serum albumin hydrosol-mediated formation of Ag3PO4/reduced graphene oxide composites for visible-light degradation of Rhodamine B solution.
J Colloid Interface Sci
PUBLISHED: 01-11-2014
Show Abstract
Hide Abstract
A cost-effective Ag(I)-bovine serum albumin (BSA) supramolecular hydrosol strategy was utilized to assemble Ag3PO4 nanospheres onto reduced graphene oxide (rGO) sheets. The obtained composites were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, UV-vis absorption spectroscopy and Fourier transform infrared spectroscopy. Compared with the pure Ag3PO4 crystals and Ag3PO4 particles prepared with Ag(I)-BSA hydrosol as precursor, the Ag3PO4/rGO composites obtained with different content of graphene oxide indicated improved visible-light-driven photocatalysis activity for the decomposition of Rhodamine B aqueous solution. The results pointed to the possibility of synthesizing graphene-based photocatalysts by metal ion-BSA hydrosol.
Related JoVE Video
ADAMTS-7 expression increases in the early stage of angiotensin II-induced renal injury in elderly mice.
Kidney Blood Press. Res.
PUBLISHED: 01-10-2014
Show Abstract
Hide Abstract
We investigated the recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), and matrix metalloproteinases (MMPs) as inflammatory mediators in inflammatory kidney damage by studying ADAMTS-1, -4, and -7 and MMP-9 expression in elderly mouse kidneys after angiotensin II (Ang II) administration.
Related JoVE Video
Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57.
Mol. Immunol.
PUBLISHED: 01-04-2014
Show Abstract
Hide Abstract
Rabies virus (RV) causes a fatal infectious disease requiring efficient post-exposure prophylaxis (PEP), which includes a rabies vaccine and rabies immunoglobulin (RIG). The single-chain antibody variable fragment (scFv), a small engineered antibody fragment derived from an antibody variable heavy chain and light chain, has the potential to replace the current application of RIG. In previous studies, we constructed and evaluated an anti-rabies virus G protein scFv (FV57) based on the monoclonal antibody CR57. Of the five cysteines in FV57, four are linked in intra-chain disulfide bonds (Cys-VH28/Cys-VH98 and Cys-VL16/Cys-VL84), and one is free (Cys-VL85). However, the thiol in Cys-VL85 neighboring Cys-VL84 in the CDR3 of the light chain is likely to mismatch with the thiol in Cys-VL16 during the renaturing process. In order to study effects of the mismatched disulfide bond, Cys-VL85 and Cys-VL84 of FV57 were mutated to serine to construct mutants FV57(VL85S) and FV57(VL84S). Furthermore, the disulfide bonds in the light chain of FV57, FV57(VL85S) and FV57(VL84S) were deleted by mutating Cys-VL16 to serine. All mutants were prepared and evaluated along with the original FV57. The results indicated that the mismatched disulfide bond of FV57 linking the light chain FR1 and CDR3 would confer deleterious negative effects on its activity against RV, likely due to spatial hindrance in the light chain CDR3. Moreover, avoidance of the disulfide bond mismatch provided an additional 30% protective efficacy against RV infection in the mouse RV challenge model. Thus, modifications of FV57 to eliminate the disulfide bond mismatch may provide a candidate therapeutic agent for effective PEP against rabies.
Related JoVE Video
Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.
Related JoVE Video
Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.
Related JoVE Video
A fiber-modified adenovirus co-expressing HSV-TK and Coli.NTR enhances antitumor activities in breast cancer cells.
Int J Clin Exp Pathol
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Breast cancers especially in late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Suicide gene therapy based on adenoviral technology will be promising strategies for such advanced diseases. We previously showed that co-expression of herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli nitroreductase (Coli.NTR) by an hTERT-driven adenovirus vector resulted in additive anti-tumor effects in breast cancer cells in vitro and in vivo. As many tumor tissue and cancer cells express low level of coxsackie-adenovirus receptor (CAR), which is the functional receptor for the fiber protein of human adenovirus serotype 5 (Ad5), novel Ad5 vectors containing genetically modifi ed fiber are attractive vehicles for achieving targeted gene transfer and improving suicide gene expression in these cancer cells. In the present study, we first built a simplified Ad5 vector platform for fiber modification and quick detection for gene transfer. Then a fiber-modified adenovirus vector containing an RGD motif in the HI loop of the fiber knob was constructed. After recombined with HSV-TK and Coli.NTR gene, this fiber-modified Ad5 vector (Ad-RGD-hT-TK/NTR) was compared with that of our previously constructed Ad5 vector (Ad-hT-TK/NTR) for its therapeutic effects in human breast cancer cell lines. The anti-tumor activity of Ad-RGD-hT-TK/NTR was significantly enhanced compared with Ad-hT-TK/NTR both in vitro and in vivo. This new vector platform provided a robust and simplified approach for capsid modification, and the fiber-modified Ad5 with double suicide genes under the control of hTERT promoter would be a useful gene therapy strategy for breast cancer.
Related JoVE Video
Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.
Related JoVE Video
Hepcidin and sports anemia.
Cell Biosci
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Iron is an important mineral element used by the body in a variety of metabolic and physiologic processes. These processes are highly active when the body is undergoing physical exercises. Prevalence of exercise-induced iron deficiency anemia (also known as sports anemia) is notably high in athletic populations, particularly those with heavy training loads. The pathogenesis of sports anemia is closely related to disorders of iron metabolism, and a more comprehensive understanding of the mechanism of iron metabolism in the course of physical exercises could expand ways of treatment and prevention of sports anemia. In recent years, there have been remarkable research advances regarding the molecular mechanisms underlying changes of iron metabolism in response to physical exercises. This review has covered these advances, including effects of exercise on duodenum iron absorption, serum iron status, iron distribution in organs, erythropoiesis, and hepcidin's function and its regulation. New methods for the treatment of exercise-induced iron deficiency are also discussed.
Related JoVE Video
[Effect of neonatal immunization with different dosage allergen on the immunity of mice when grown up].
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi
PUBLISHED: 12-10-2013
Show Abstract
Hide Abstract
To study the effect of neonatal immunization with different dosage allergen on the immunity of mice when grown.
Related JoVE Video
Intermedin inhibits macrophage foam-cell formation via tristetraprolin-mediated decay of CD36 mRNA.
Cardiovasc. Res.
PUBLISHED: 11-18-2013
Show Abstract
Hide Abstract
CD36-mediated uptake of oxidized low-density lipoprotein (oxLDL) plays a pivotal role in macrophage foam-cell formation and atherogenesis. Previously we reported on intermedin (IMD), a novel member of the calcitonin gene-related peptide family, in atherosclerotic plaque reducing atherogenesis in apolipoprotein E-deficient (apoE(-/-)) mice. Here, we studied the role of IMD in CD36-mediated macrophage foam-cell formation.
Related JoVE Video
Antitumor effect of adenoviral vector prime protein boost immunity targeting the MUC1 VNTRs.
Oncol. Rep.
PUBLISHED: 10-17-2013
Show Abstract
Hide Abstract
Mucin 1 (MUC1) is a tumor-associated antigen that is overexpressed in several adenocarcinomas. However, clinical trials with MUC1 showed that MUC1 is a relatively poor immunogen in humans. In view of the low immunogenicity of this protein vaccine, we designed a method based on an immunoadjuvant and immunization strategy to enhance the cellular immune response to this protein vaccine. DDA/MPL has been evaluated as an adjuvant to induce strong immunity for the tuberculosis vaccine. However, its adjuvant role combined with the vaccine targeting MUC1 in malignant carcinomas has not previously been reported. Our previous study showed that adenovirus prime protein boost vaccination could significantly enhance the cellular immunity and antitumor efficacy. In our study, we used MUC1 VNTRs as the target of cancer vaccine and DDA/MPL as the adjuvant to enhancing the cellular immunity of recombinant MUC1 protein vaccine, and an AD-9M adenoviral vector prime-recombinant protein and DDA/MPL boost (designated MUC-1 VPP vaccine) strategy was studied to enhance the antitumor efficacy. The results demonstrated that antigen-specific IFN-?-secreting T cells were increased by 2-fold, and cytotoxic T lymphocytes (CTLs) were induced effectively when the protein vaccine was combined with the DDA/MPL adjuvant. Moreover, the vaccination induced nearly 60% inhibition of the growth of B16 melanoma in mice and prolonged the survival of tumor-bearing mice. The inhibition was correlated with the specific immune responses induced by the MUC1 VPP vaccine. The data suggested that DDA/MPL-adjuvant MUC-1 VPP vaccine may be developed into effective tumor vaccines for melanomas and possibly for other tumors expressing MUC1 protein.
Related JoVE Video
Preparation and diagnostic use of a novel recombinant single-chain antibody against rabies virus glycoprotein.
Appl. Microbiol. Biotechnol.
PUBLISHED: 09-06-2013
Show Abstract
Hide Abstract
Rabies virus (RABV) causes a fatal infectious disease, but effective protection may be achieved with the use of rabies immunoglobulin and a rabies vaccine. Virus-neutralizing antibodies (VNA), which play an important role in the prevention of rabies, are commonly evaluated by the RABV neutralizing test. For determining serum VNA levels or virus titers during the RABV vaccine manufacturing process, reliability of the assay method is highly important and mainly dependent on the diagnostic antibody. Most diagnostic antibodies are monoclonal antibodies (mAbs) made from hybridoma cell lines and are costly and time consuming to prepare. Thus, production of a cost-effective mAb for determining rabies VNA levels or RABV titers is needed. In this report, we describe the prokaryotic production of a RABV-specific single-chain variable fragment (scFv) protein with a His-tag (scFv98H) from a previously constructed plasmid in a bioreactor, including the purification and refolding process as well as the functional testing of the protein. The antigen-specific binding characteristics, affinity, and relative affinity of the purified protein were tested. The scFv98H antibody was compared with a commercial RABV nucleoprotein mAb for assaying the VNA level of anti-rabies serum samples from different sources or testing the growth kinetics of RABV strains for vaccine manufactured in China. The results indicated that scFv98H may be used as a novel diagnostic tool to assay VNA levels or virus titers and may be used as an alternative for the diagnostic antibody presently employed for these purposes.
Related JoVE Video
The regulation of iron metabolism in the mononuclear phagocyte system.
Expert Rev Hematol
PUBLISHED: 09-03-2013
Show Abstract
Hide Abstract
The daily iron absorption and loss are small and iron metabolism in human is characterized by a limited external exchange and by an efficient reutilization of iron from internal sources. The mononuclear phagocyte system (MPS) plays a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes, which is important in maintaining iron homeostasis. Many iron-related proteins are expressed in the MPS, including heme oxygenase (HO) for heme degradation, the iron importer transferrin receptor 1 (TfR1) and divalent metal transport 1 (DMT1), the iron exporter ferroportin 1 (FPN1) and the iron regulatory hormone hepcidin. Insights into the regulatory mechanisms that control the regulation of iron metabolism proteins in the MPS will deepen our understanding about the molecular mechanism of iron homeostasis and iron-related diseases.
Related JoVE Video
The changes in mGluR2 and mGluR7 expression in rat medial vestibular nucleus and flocculus following unilateral labyrinthectomy.
Int J Mol Sci
PUBLISHED: 08-21-2013
Show Abstract
Hide Abstract
It is known that the medial vestibular nucleus (MVN) and the cerebellar flocculus are the key areas, which contribute to the behavioral recovery ("vestibular compensation") after unilateral labyrinthectomy (UL). In these areas, how the genetic activities of the metabotropic glutamate receptors mGluR2 and mGluR7 performance after UL is unknown. With the means of quantitative real-time PCR, Western blotting, and immunohistochemistry, we analyzed the expression of mGluR2 and mGluR7 in the bilateral MVN and the flocculus of rats in different stages after UL (the 1st, 3rd, and 7th day). Our results show that in the MVN, the mRNA, and protein expressions of mGluR7 were ipsilaterally decreased at the 1st day following UL. However, in the MVN, no change was observed in the mRNA and protein expressions of mGluR2. On the other hand, the mRNA and protein expression of mGluR2 were enhanced in the ipsilateral flocculus at the 1st day following UL, while in the flocculus no change was shown in mGluR7 mRNA and protein expressions. Our results suggest that mGluR2 and mGluR7 may contribute to the early rebalancing of spontaneous resting activity in the MVN.
Related JoVE Video
UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-08-2013
Show Abstract
Hide Abstract
At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.
Related JoVE Video
Pd-catalyzed addition of boronic acids to ynol ethers: a highly regio- and stereoselective synthesis of trisubstituted vinyl ethers.
Chem. Commun. (Camb.)
PUBLISHED: 07-22-2013
Show Abstract
Hide Abstract
A Pd-catalyzed addition of boronic acids to ynol ethers has been realized, delivering trisubstituted vinyl ethers in good yields with perfect control of the regio- and stereoselectivity. The reaction proceeds under mild conditions and exhibits excellent functional group compatibility. Moreover, the resultant products can be converted into pentasubstituted benzenes via the tandem Diels-Alder/aromatization reaction.
Related JoVE Video
Sphere Formation Assay is Not an Effective Method for Cancer Stem Cell Derivation and Characterization from the Caco-2 Colorectal Cell Line.
Curr Stem Cell Res Ther
PUBLISHED: 07-09-2013
Show Abstract
Hide Abstract
Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.
Related JoVE Video
Development of a sensitive and reliable high performance liquid chromatography method with fluorescence detection for high-throughput analysis of multi-class mycotoxins in Coix seed.
Anal. Chim. Acta
PUBLISHED: 06-26-2013
Show Abstract
Hide Abstract
As an edible and medicinal plant, Coix seed is readily contaminated by more than one group of mycotoxins resulting in potential risk to human health. A reliable and sensitive method has been developed to determine seven mycotoxins (aflatoxins B1, B2, G1, G2, zearalenone, ?-zearalenol, and ?-zearalenol) simultaneously in 10 batches of Coix seed marketed in China. The method is based on a rapid ultrasound-assisted solid-liquid extraction (USLE) using methanol/water (80/20) followed by immunoaffinity column (IAC) clean-up, on-line photochemical derivatization (PCD), and high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). Careful optimization of extraction, clean-up, separation and detection conditions was accomplished to increase sample throughput and to attain rapid separation and sensitive detection. Method validation was performed by analyzing samples spiked at three different concentrations for the seven mycotoxins. Recoveries were from 73.5% to 107.3%, with relative standard deviations (RSDs) lower than 7.7%. The intra- and inter-day precisions, expressed as RSDs, were lower than 4% for all studied analytes. Limits of detection and quantification ranged from 0.01 to 50.2 ?g kg(-1), and from 0.04 to 125.5 ?g kg(-1), respectively, which were below the tolerance levels for mycotoxins set by the European Union. Samples that tested positive were further analyzed by HPLC tandem electrospray ionization mass spectrometry for confirmatory purposes. This is the first application of USLE-IAC-HPLC-PCD-FLD for detecting the occurrence of multi-class mycotoxins in Coix seed.
Related JoVE Video
Deficiency of cartilage oligomeric matrix protein causes dilated cardiomyopathy.
Basic Res. Cardiol.
PUBLISHED: 06-17-2013
Show Abstract
Hide Abstract
Alterations in cardiac extracellular matrix are involved in dilated cardiomyopathy (DCM) and its progression to heart failure. The matricellular protein cartilage oligomeric matrix protein (COMP) has been indicated localized in the heart. However, the role of COMP in cardiac homeostasis and disease remains elusive. COMP (-/-) mice, both male and female, developed DCM spontaneously at young age (3-5 months), with impaired cardiac function. Assessment of postnatal COMP (-/-) heart at 1 month, although functionally normal, revealed severe cardiac ultrastructure defect, in parallel with cardiomyocyte apoptosis, myofilament loss, connexin-43 deficiency and matrix metalloproteinase activation. Decreased COMP expression was observed in the heart sample of DCM patients compared with donor heart. Mechanistically, COMP (-/-) heart exhibited reduced integrin ?1 expression and signaling. Ectopic expression of COMP or integrin ?1 rescued COMP-deficiency-induced cardiomyocyte apoptosis, myofilament dissolution, and connexin-43 aberrance. Additionally, COMP directly bonded to the extracellular ?-tail domain of integrin ?1, prevented integrin ?1 ubiquitination/degradation, and maintained the cardiac homeostasis. COMP-integrin ?1 axis is a potential target of DCM.
Related JoVE Video
A novel modified peptide derived from membrane-proximal external region of human immunodeficiency virus type 1 envelope significantly enhances retrovirus infection.
J. Pept. Sci.
PUBLISHED: 06-11-2013
Show Abstract
Hide Abstract
Efficient gene transfer is a critical goal in retroviral transduction. Several peptides capable of forming amyloid fibrils, such as the 39-residue semen-derived infection-enhancing peptide (SEVI), have demonstrated the ability to boost retroviral gene delivery. Here, a 13-residue peptide P13 (Ac-(671) NWFDITNWLWYIK(683) ) derived from the membrane-proximal external region of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane protein, together with its 16-residue peptide derivative (P16) were found to enhance HIV-1 infection significantly. Both peptides, P13 and P16, could form amyloid fibril structures to potently enhance HIV-1 infectivity. Further investigations showed that both aromatic Trp residues and cationic Lys residues contributed to the enhancement of HIV-1 infection by these two active peptides. P16 could more effectively augment HIV-1 YU-2 infection than SEVI, implying its potential applications as a tool in the lab to improve gene transfer rates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Related JoVE Video
Research on the practical parameters of sex pheromone traps for the oriental fruit moth.
Pest Manag. Sci.
PUBLISHED: 05-28-2013
Show Abstract
Hide Abstract
The oriental fruit moth (OFM) is a worldwide fruit-boring insect pest. In China, OFM monitoring traps use a sex pheromone lure, but their overall design is varied. As such, there is a critical need to develop a standardised OFM trap design. In this field study, ten different trap shapes in varying combinations of colours and sizes (such as trap length and surface area) were examined.
Related JoVE Video
Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication.
World J. Gastroenterol.
PUBLISHED: 05-20-2013
Show Abstract
Hide Abstract
To investigate the effects of suberoylanilide hydroxamic acid (SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line (HepG2.2.15) and hepatitis B virus (HBV) replication.
Related JoVE Video
Purification of eukaryotic tetherin/Vpu proteins and detection of their interaction by ELISA.
Protein Expr. Purif.
PUBLISHED: 05-15-2013
Show Abstract
Hide Abstract
Tetherin/BST-2/CD317 inhibits HIV-1 release from infected cells, while HIV-1 Vpu efficiently antagonizes tetherin based on intermolecular interactions between the transmembrane domains of each protein. In this study, we successfully partially purified His-tagged tetherin with a glycophosphatidylinositol deletion (delGPI) and His-tagged full-length Vpu from transiently transfected 293T cells using affinity chromatography. The in vitro interaction between these purified proteins was observed by a pull-down assay and ELISA. Detection of the Vpu/tetherin interaction by ELISA is a novel approach that would be advantageous for inhibitor screening in vitro. Successful co-purification of the tetherin/Vpu complex also provides a basis for further structural studies.
Related JoVE Video
Genotype variations in cadmium and lead accumulations of leafy lettuce (Lactuca sativa L.) and screening for pollution-safe cultivars for food safety.
Environ Sci Process Impacts
PUBLISHED: 05-10-2013
Show Abstract
Hide Abstract
Heavy-metals in polluted soils can accumulate in plants and threaten crop safety. To evaluate the risk of heavy-metal pollution in leafy lettuce (Lactuca sativa L.), two pot experiments were conducted to investigate Cd and Pb accumulation and transfer potential in 28 cultivars of lettuce and to screen for low-Cd and low-Pb accumulative cultivars. In the three treatments, 5.2-fold, 4.8-fold and 4.8-fold differences in the shoot Cd concentration were observed between the cultivars with the highest and the lowest Cd concentrations, respectively. This genotype variation was sufficiently large to identify low-Cd accumulative genotypes to reduce Cd contamination in food. Cadmium accumulation in the low-Cd accumulative genotypes was significantly positively correlated with Pb accumulation. At the cultivar level, Cd and Pb accumulation in lettuce was stable and genotype-dependent. High Pb soil levels did not affect shoot Cd accumulation in lettuce. Lettuce was concluded to be at high risk for Cd pollution and low risk for Pb pollution. Among the tested cultivars, cvs. SJGT, YLGC, N518, and KR17 had the lowest Cd and Pb accumulation abilities in shoots and are thus important parental material for breeding pollution-safe cultivars to minimize Cd and Pb accumulation.
Related JoVE Video
Low-pH rescue of acid-sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-regulated arginine decarboxylase system.
J. Bacteriol.
PUBLISHED: 05-03-2013
Show Abstract
Hide Abstract
For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ?aroD and ?phoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.
Related JoVE Video
A novel rodent model of posterior ischemic optic neuropathy.
JAMA Ophthalmol
PUBLISHED: 04-02-2013
Show Abstract
Hide Abstract
To develop a reliable, reproducible rat model of posterior ischemic optic neuropathy (PION) and study the cellular responses in the optic nerve and retina.
Related JoVE Video
Simultaneous multi-mycotoxin determination in nutmeg by ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection.
Analyst
PUBLISHED: 03-15-2013
Show Abstract
Hide Abstract
A simple and sensitive analytical method based on ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with high performance liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection (USLE-IAC-HPLC-PCD-FLD) has been developed for simultaneous multi-mycotoxin determination of aflatoxins B1, B2, G1, G2 (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) in 13 edible and medicinal nutmeg samples marketed in China. AFs and OTA were extracted from nutmeg samples by ultrasonication using a methanol?:?water (80?:?20, v/v) solution, followed by an IAC clean-up step. Different USL extraction conditions, pre-processing ways for nutmeg sample and clean-up columns for mycotoxins, as well as HPLC-PCD-FLD parameters (mobile phase, column temperature, elution procedure, excitation and emission wavelengths) were optimized. This method, which was appraised for analyzing nutmeg samples, showed satisfactory results with reference to limits of detection (LODs) (from 0.02 to 0.25 ?g kg(-1)), limits of quantification (LOQs) (from 0.06 to 0.8 ?g kg(-1)), linear ranges (up to 30 ng mL(-1) for AFB1, AFG1 and OTA and 9 ng mL(-1) for AFB2 and AFG2), intra- and inter-day variability (all <2%) and average recoveries (from 79.6 to 90.8% for AFs and from 93.6 to 97.3% for OTA, respectively). The results of the application of developed method in nutmeg samples have elucidated that four samples were detected with contamination of AFs and one with OTA. AFB1 was the most frequently found mycotoxin in 30.8% of nutmeg samples at contamination levels of 0.73-16.31 ?g kg(-1). At least two different mycotoxins were co-occurred in three samples, and three AFs were simultaneously detected in one sample.
Related JoVE Video
Short-Form CDYLb but not long-form CDYLa functions cooperatively with histone methyltransferase G9a in hepatocellular carcinomas.
Genes Chromosomes Cancer
PUBLISHED: 03-14-2013
Show Abstract
Hide Abstract
In hepatocellular carcinomas (HCCs), the levels of histone H3 dimethylation at lysine 9 (H3K9me2) and its corresponding histone methyltransferase G9a are significantly elevated. Recently, G9a was reported to form a complex with the H3K9 methylation effector protein CDYL, but little is known about the expression of CDYL in HCC patients. The human CDYL gene produces two transcripts, a long form (CDYLa) and a short form (CDYLb), but it is unclear whether the protein products have different functions. The aim of this study was to investigate the distinctions between CDYLa and CDYLb and their expression levels in HCC tissues. We first examined binding abilities of the different CDYL forms with methylated H3 peptides by a pull-down assay. Human CDYLb (h-CDYLb) specifically recognized H3Kc9me2 and H3Kc9me3 modifications, whereas human CDYLa (h-CDYLa) did not interact with any methylated H3 peptides. Similarly, mouse CDYLb (m-CDYLb) specifically bound with di- and tri-methylated H3Kc9 peptides, while mouse CDYLa (m-CDYLa) lacked that ability. Affinity purification also was used to identify the distinct composition of the h-CDYLa or h-CDYLb protein complex. h-CDYLb was found in a multiprotein complex with G9a and GLP, while the h-CDYLa complex did not contain these two enzymes. Consistent with the protein complex composition, h-CDYLb and G9a were both upregulated in HCC tissues, compared with adjacent non-cancerous liver tissues. Furthermore, the positive correlation between expression levels of h-CDYLb and G9a was statistically significant. In contrast, h-CDYLa showed no enrichment in HCC tissues. These findings suggest that h-CDYLb and G9a are cooperatively involved in HCC.
Related JoVE Video
Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-? binding to Vif.
Retrovirology
PUBLISHED: 03-04-2013
Show Abstract
Hide Abstract
The HIV-1 accessory factor Vif is necessary for efficient viral infection in non-permissive cells. Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3 proteins that confer the non-permissive phenotype by tethering them (APOBEC3DE/3F/3G) to the Vif-CBF-?-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-?-EloB-EloC-Cul5-Rbx) E3 complex to induce their proteasomal degradation. EloB and EloC were initially reported as positive regulatory subunits of the Elongin (SIII) complex. Thereafter, EloB and EloC were found to be components of Cul-E3 complexes, contributing to proteasomal degradation of specific substrates. CBF-? is a newly identified key regulator of Vif function, and more information is needed to further clarify its regulatory mechanism. Here, we comprehensively investigated the functions of EloB (together with EloC) in the Vif-CBF-?-Cul5 E3 ligase complex.
Related JoVE Video
Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.
J. Biosci. Bioeng.
PUBLISHED: 02-28-2013
Show Abstract
Hide Abstract
The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 ?g/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 ?g/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.
Related JoVE Video
The novel replication-defective vaccinia virus (Tiantan strain)-based hepatitis C virus vaccine induces robust immunity in macaques.
Mol. Ther.
PUBLISHED: 02-25-2013
Show Abstract
Hide Abstract
The induction of a robust neutralizing antibody (nAb) response is likely to be as essential as specific cell-mediated immunity (CMI) against multiple antigens for the development of effective preventive and therapeutic vaccines against hepatitis C virus (HCV) infection in humans. To date, no data on the immunogenicity of the replication-defective vaccinia virus (derived from the Tiantan strain) (rNTV)-based HCV vaccine in primates have been reported. This study describes in detail the immunogenicity of various vaccine candidates in rhesus macaques, including rNTV-based and replication-defective recombinant adenoviral (rAd)-based HCV vaccines, as well as HCV pseudotyped virus-like particles (HCVpp). Our data showed that rAd-HCV vaccine boosting induced robust CMI, while priming or boosting with HCVpp enhanced the antigen-specific nAb response after rAd-HCV vaccination; however, CMI was not enhanced. Vaccination includes rNTV-HCV priming induced robust antigen-specific antibody, particularly nAbs, and CMI responses. Furthermore, more robust and longer-lasting CMI and higher cytokine levels (both Th1 and Th2 types, especially IFN-?) resulted from boosting with rAd-HCV. We conclude that the rNTV-based HCV vaccine induces robust nAbs and CMI when combined with a heterogeneous primer-booster strategy, which shows promise for development of a human HCV vaccine.
Related JoVE Video
Hyperhomocysteinemia promotes insulin resistance by inducing endoplasmic reticulum stress in adipose tissue.
J. Biol. Chem.
PUBLISHED: 02-17-2013
Show Abstract
Hide Abstract
Type 2 diabetes is a chronic inflammatory metabolic disease, the key point being insulin resistance. Endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of type 2 diabetes. Previously, we found that hyperhomocysteinemia (HHcy) induced insulin resistance in adipose tissue. Here, we hypothesized that HHcy induces ER stress, which in turn promotes insulin resistance. In the present study, the direct effect of Hcy on adipose ER stress was investigated by the use of primary rat adipocytes in vitro and mice with HHcy in vivo. The mechanism and the effect of G protein-coupled receptor 120 (GPR120) were also investigated. We found that phosphorylation or expression of variant ER stress markers was elevated in adipose tissue of HHcy mice. HHcy activated c-Jun N-terminal kinase (JNK), the downstream signal of ER stress in adipose tissue, and activated JNK participated in insulin resistance by inhibiting Akt activation. Furthermore, JNK activated c-Jun and p65, which in turn triggered the transcription of proinflammatory cytokines. Both in vivo and in vitro assays revealed that Hcy-promoted macrophage infiltration aggravated ER stress in adipose tissue. Chemical chaperones PBA and TUDCA could reverse Hcy-induced inflammation and restore insulin-stimulated glucose uptake and Akt activation. Activation of GPR120 reversed Hcy-induced JNK activation and prevented inflammation but not ER stress. Therefore, HHcy inhibited insulin sensitivity in adipose tissue by inducing ER stress, activating JNK to promote proinflammatory cytokine production and facilitating macrophage infiltration. These findings reveal a new mechanism of HHcy in the pathogenesis of insulin resistance.
Related JoVE Video
Methylselenol prodrug enhances MDV3100 efficacy for treatment of castration-resistant prostate cancer.
Int. J. Cancer
PUBLISHED: 02-14-2013
Show Abstract
Hide Abstract
The next-generation antiandrogen MDV3100 prolongs overall survival of patients with metastatic castration-resistant prostate cancer (CRPC). However, patient responses are variable, and survival benefit remains relatively small. Developing effective modality to improve MDV3100 efficacy is urgently needed. Recent evidence suggests that constitutively active androgen receptor splice variants (AR-Vs) drive resistance to MDV3100. In our study, we show that methylselenol prodrug downregulates the expression and activity of both the full-length AR (AR-FL) and AR-Vs. The downregulation is independent of androgen and could be attributable to repressed transcription of the AR gene. Cotreatment with methylselenol prodrug and MDV3100 suppresses AR signaling more dramatically than either agent alone, and synergistically inhibits the growth of CRPC cells in vitro. The combinatorial efficacy is observed in not only AR-V-expressing cells but also cells expressing predominantly AR-FL, likely owing to the ability of the two drugs to block the AR signaling cascade at distinct steps. Ectopic expression of AR-FL or AR-V7 attenuates the combinatorial efficacy, indicating that downregulating AR-FL and AR-V7 is importantly involved in mediating the combinatorial efficacy. Significantly, methylselenol prodrug also downregulates AR-FL and AR-Vs in vivo and substantially improves the antitumor efficacy of MDV3100. These findings support a potential combination therapy for improving MDV3100 efficacy, and provide a rationale for evaluating the clinical application of combining methylselenol prodrug with MDV3100 for the treatment of CRPC.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.