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Articles by Hongbo Guo in JoVE

 JoVE Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 2Deparment of Biochemistry, Research and Innovation Centre, University of Regina, 3Department of Medical Genetics and Microbiology, University of Toronto


JoVE 4057

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Other articles by Hongbo Guo on PubMed

Laminated and Functionally Graded Hydroxyapatite/yttria Stabilized Tetragonal Zirconia Composites Fabricated by Spark Plasma Sintering

Hydroxyapatite (HA) ceramics have been conventionally strengthened and toughened in the form of composites and coatings. New microstructural designs and processing methodologies are still needed for the improvement of the mechanical properties of HA-based ceramics. This study was to prepare laminated and functionally graded HA/yttria stabilized tetragonal zirconia (Y-TZP) composites by the relatively new process of spark plasma sintering (SPS). The microstructure and the mechanical properties of the laminated and functionally graded composites were studied for possible orthopedic applications. It was found that the laminated and functionally graded HA/Y-TZP composites could be densified at 1200 degrees C within 5 min by the SPS process and the average HA grain size in the composite layers was reduced by half due to the well-dispersed Y-TZP second phase. The HA phase in the composite layers was stable up to 1200 degrees C and the Y-TZP second phase remained the tetragonal zirconia (t-ZrO(2)) phase after being processed at the highest temperature of 1250 degrees C. The laminated and functionally graded HA/Y-TZP composites exhibited much improved mechanical properties compared with the pure HA ceramics; the bending strength of the composites reached about 200 MPa, double the strength of the pure HA ceramics.

Additional Value of PET/CT over PET in Assessment of Locoregional Lymph Nodes in Thoracic Esophageal Squamous Cell Cancer

The aim of this study was to compare the value of reviewing combined 18F-FDG PET/CT images with that of reviewing side-by-side PET and CT images in the diagnosis of locoregional lymph node metastases in patients with esophageal squamous cell cancer.

Serial Hypoxia Imaging with 99mTc-HL91 SPECT to Predict Radiotherapy Response in Nonsmall Cell Lung Cancer

To evaluate the relationship between 99mTc-HL91 (99mTc labeled 4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime) SPECT (single photon emission computed tomography) hypoxia imaging and the treatment outcome and also to assess changes in tumor oxygenation during the course of radiotherapy.

Diagnostic and Prognostic Value of 18F-FDG PET/CT for Patients with Suspected Recurrence from Squamous Cell Carcinoma of the Esophagus

Patients with esophageal squamous cell carcinoma (ESCC) are commonly at high risk of recurrence within 2 y after initial treatment. The aim of this study was to evaluate the role of 18F-FDG PET/CT in patients with possibly recurrent ESCC who underwent definitive treatment.

Upregulation of Survivin by Leptin/STAT3 Signaling in MCF-7 Cells

Leptin and its receptors are overexpressed in breast cancer tissues and correlate with poor prognosis. Survivin, a member of the inhibitor of apoptosis protein (IAP) gene family, is generally upregulated in tumor tissues and prevents tumor cells from apoptosis. Here we showed that leptin upregulated survivin mRNA and protein expression in MCF-7 breast cancer cells. Meanwhile, leptin suppressed docetaxel-induced apoptosis by inhibiting caspase activity. Knockdown of signal transducer and activator transcription 3 (STAT3) expression by small interfering RNA (siRNA) blocked leptin-induced upregulation of survivin. TransAM ELISA showed that leptin increased nuclear translocation of active STAT3. In addition, chromatin immunoprecipitation (ChIP) assay detected an enhanced binding of STAT3 to survivin promoter in MCF-7 cells after treatment by leptin. Further studies showed that leptin enhanced the transcriptional activity of survivin promoter. Collectively, our findings identify leptin/STAT3 signaling as a novel pathway for survivin expression in breast cancer cells.

Dual-time-point FDG PET for the Evaluation of Locoregional Lymph Nodes in Thoracic Esophageal Squamous Cell Cancer

Compare dual-time-point and single-time 18F-FDG PET in the evaluation of locoregional lymph node metastases in patients with esophageal squamous cell cancer. Then assess the efficacy of dual-time-point PET in the diagnosis of such nodules.

ATP-sensitive Potassium Channels Control Glioma Cells Proliferation by Regulating ERK Activity

Ion channels are found in a variety of cancer cells and necessary for cell cycle and cell proliferation. The roles of K(+) channels in the process are, however, poorly understood. In the present study, we report that adenosine triphosphate (ATP)-sensitive potassium channel activity plays a critical role in the proliferation of glioma cells. The expression of K(ATP) channels in glioma tissues was greatly increased than that in normal tissues. Treatment of glioma cells with tolbutamide, K(ATP) channels inhibitor, suppressed the proliferation of glioma cells and blocked glioma cell cycle in G(0)/G(1) phase. Similarly, downregulation of K(ATP) channels by small interfering RNA (siRNA) inhibited glioma cell proliferation. On the other hand, K(ATP) channels agonist diazoxide and overexpression of K(ATP) channels promoted the proliferation of glioma cells. Moreover, inhibiting K(ATP) channels slowed the formation of tumor in nude mice generated by injection of glioma cells. Whereas activating K(ATP) channels promoted development of tumor in vivo. The effect of K(ATP) channels activity on glioma cells proliferation is mediated by extracellular signal-regulated kinase (ERK) activation. We found that activating K(ATP) channel triggered ERK activation and inhibiting K(ATP) channel depressed ERK activation. U-0126, the mitogen-activated protein kinase kinase (MAPK kinase) inhibitors blocked ERK activation and cell proliferation induced by diazoxide. Furthermore, constitutively activated MEK plasmids transfection reversed the inhibitory effects of tolbutamide on glioma proliferation, lending further support for a role of ERK in mediating this process. Our results suggest that K(ATP) channels control glioma cell proliferation via regulating ERK pathway. We concluded that K(ATP) channels are important in pathological cell proliferation and open a promising pathway for novel targeted therapies.

Identification of Direct Target Engagement Biomarkers for Kinase-targeted Therapeutics

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3'-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1(Ser410) and p-PDK1(Thr513) are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1(Ser241)), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

Control of the RNA Polymerase II Phosphorylation State in Promoter Regions by CTD Interaction Domain-containing Proteins RPRD1A and RPRD1B

RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.

Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake

Delivery and toxicity are critical issues facing nanomedicine research. Currently, there is limited understanding and connection between the physicochemical properties of a nanomaterial and its interactions with a physiological system. As a result, it remains unclear how to optimally synthesize and chemically modify nanomaterials for in vivo applications. It has been suggested that the physicochemical properties of a nanomaterial after synthesis, known as its "synthetic identity", are not what a cell encounters in vivo. Adsorption of blood components and interactions with phagocytes can modify the size, aggregation state, and interfacial composition of a nanomaterial, giving it a distinct "biological identity". Here, we investigate the role of size and surface chemistry in mediating serum protein adsorption to gold nanoparticles and their subsequent uptake by macrophages. Using label-free liquid chromatography tandem mass spectrometry, we find that over 70 different serum proteins are heterogeneously adsorbed to the surface of gold nanoparticles. The relative density of each of these adsorbed proteins depends on nanoparticle size and poly(ethylene glycol) grafting density. Variations in serum protein adsorption correlate with differences in the mechanism and efficiency of nanoparticle uptake by a macrophage cell line. Macrophages contribute to the poor efficiency of nanomaterial delivery into diseased tissues, redistribution of nanomaterials within the body, and potential toxicity. This study establishes principles for the rational design of clinically useful nanomaterials.

Anti-miR-155 Oligonucleotide Enhances Chemosensitivity of U251 Cell to Taxol by Inducing Apoptosis

The oncogene, microRNA-155, is significantly elevated in GBM (glioblastoma multiforme), regulating multiple genes associated with cancer cell proliferation, apoptosis and invasiveness. Thus, miR-155 can theoretically become a target for enhancement of the chemotherapy in cancer. Down-regulating miR-155 to enhance the effect of taxol has not been studied in human GBM. Human GBM U251 cells were treated with taxol and the miR-155 inhibitor alone or in combination. IC50 values were dramatically decreased in cells treated with miR-155 inhibitor combined with taxol, to a greater extent than those treated with taxol alone. Furthermore, the miR-155 inhibitor significantly enhanced apoptosis in U251 cells. The data suggest that miR-155 blockage increased the chemosensitivity to taxol in GBM cells, making combined treatment an effective therapeutic strategy for controlling the growth by inhibiting EAG1 expression.

Genetic Diversity of Avian Infectious Bronchitis Coronavirus in Recent Years in China

Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2010, and they were genotyped by comparison with 19 reference strains in the present study. The results showed that LX4-type isolates are still the predominant IBVs circulating in chicken flocks in China, and these isolates could be grouped further into two clusters. Viruses in each cluster had favored amino acid residues at different positions in the S1 subunit of the spike protein. In addition, a recombination event was observed to have occurred between LX4- and tl/CH/LDT3/03I-type strains, which contributed to the emergence of a new strain. The most important finding of the study is the isolation and identification of Taiwan II-type (TW II-type) strains of IBV in mainland China in recent years. The genome of TW II-type IBV strains isolated in mainland China has experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. Pathogenicity testing and sequence analysis of the 3' terminal untranslated region revealed that TW II-type IBV strains isolated in mainland China have a close relationship with the embryo-passaged, attenuated TW2296/95.

MiR-296-3p Regulates Cell Growth and Multi-drug Resistance of Human Glioblastoma by Targeting Ether-à-go-go (EAG1)

MicroRNAs (miRNAs) - short non-coding RNA molecules - post-transcriptionally regulate gene expressions and play crucial roles in diverse biological processes such as development, differentiation, apoptosis and proliferation. In order to investigate the possible role of miRNAs in the development of multi-drug resistance (MDR) in human glioblastoma, we first detected (by Western blotting, real-time polymerase chain reaction [RT-PCR] and immunohistochemistry) the expression of miR-296-3p and ether-à-go-go (EAG1 or KCNH1) in U251 cells, U251/imatinib mesylate (U251AR cells) and clinical specimens. The results showed that miR-296-3p was down-regulated in U251AR cells, concurrent with the up-regulation of EAG1 protein, compared with the parental U251 cell line. In vitro drug sensitivity assay demonstrated that over-expression of miR-296-3p sensitised glioblastoma (GBM) cells to anticancer drugs, whereas down-expression using antisense oligonucleotides conferred MDR. Ectopic expression of miR-296-3p reduced EAG1 expression and suppressed cell proliferation drug resistance, and the luciferase activity of an EAG1 3'-untranslated region-based reporter construct in U251AR cells, whereas EAG1 over-expression rescued the suppressive effect of miR-296-3p in U251AR cells. We also found that EAG1 was widely over-expressed and inversely correlated with miR-296-3p in clinical specimens. Taken together, our findings suggest that miR-296-3p may play a role of MDR in glioblastoma at least in part by targeting EAG1.

Seasonal Distribution and Epidemiological Characteristics of Human Metapneumovirus Infections in Pediatric Inpatients in Southeast China

Human metapneumovirus (hMPV) is an important respiratory pathogen in young children whose seasonal activity varies substantially from year to year among different populations. This study was conducted to investigate if there was a seasonal variation in the incidence of hMPV infection in young children and possible associations between hMPV infection and local meteorological parameters in Suzhou, China. A total of 6,655 children with acute respiratory tract infection (ARTI) admitted to the Children's Hospital affiliated to Soochow University, Suzhou, were tested from January 2006 to December 2009 for the presence of hMPV using reverse-transcription polymerase chain reaction. The relationship between the presence of the virus and regional meteorological conditions was analyzed by linear and multivariate regression analysis. The overall hMPV infection incidence over the four-year study was 8.2 %, 8.1 %, 12.7 % and 7.4 % per year, respectively. Four hundred eighty-eight hMPV-positive children (78.2 %) were younger than 3 years of age. hMPV infections appear to have a seasonal distribution in Suzhou. In 2006, 2007 and 2009, the peak seasons were in December to January, while in 2008, the peak of hMPV activity occurred in May. The incidence of hMPV infection was negatively correlated with the average monthly temperature and rainfall. hMPV was one of the most common viral pathogens after respiratory syncytial virus that was associated with acute respiratory tract infection in children in Suzhou. hMPV infection occurred throughout the year with peaks during late winter and early spring. Climatic factors, especially monthly average temperature, may affect the prevalence of hMPV in Suzhou.

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