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In JoVE (1)

Other Publications (20)

Articles by Li Qin Zhang in JoVE

 JoVE Biology

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

1Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City


JoVE 4393

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

Other articles by Li Qin Zhang on PubMed

Interactions Between PBEF and Oxidative Stress Proteins--a Potential New Mechanism Underlying PBEF in the Pathogenesis of Acute Lung Injury

Identification of pre-B-cell colony-enhancing factor (PBEF) interacting partners may reveal new molecular mechanisms of PBEF in the pathogenesis of acute lung injury (ALI). The interactions between PBEF and NADH dehydrogenase subunit 1(ND1), ferritin light chain and interferon induced transmembrane 3 (IFITM3) in human pulmonary vascular endothelial cells were identified and validated. ND1, ferritin and IFITM3 are involved in oxidative stress and inflammation. Overexpression of PBEF increased its interactions and intracellular oxidative stress, which can be attenuated by rotenone. The interaction modeling between PBEF and ND1 is consistent with the corresponding experimental finding. These interactions may underlie a novel role of PBEF in the pathogenesis of ALI.

Augmentation of Pulmonary Epithelial Cell IL-8 Expression and Permeability by Pre-B-cell Colony Enhancing Factor

Previous studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability.

Mogrosides Extract from Siraitia Grosvenori Scavenges Free Radicals in Vitro and Lowers Oxidative Stress, Serum Glucose, and Lipid Levels in Alloxan-induced Diabetic Mice

This study evaluated the supplementation of a mogrosides extract (MG) from fruits of Siraitia grosvenori on reducing oxidative stress, hyperglycemia, and hyperlipidemia in alloxan-induced diabetic mice. The oxygen free radical scavenging activity of MG was also assessed in vitro. After induction of diabetes, a significant increase in the levels of serum glucose, total cholesterol (TC), triacylglycerol (TG), and hepatic malondialdehyde (MDA) as well as a reduction in the level of hepatic high-density lipoprotein cholesterol (HDL-C) associated with diminution of the corresponding antioxidant enzymes, such as glutathione peroxidase (GSH-Px) and superoxide dismutase, were observed in all diabetic mice. Treatment of diabetic mice with MG (100, 300, and 500 mg/kg ) for 4 weeks significantly decreased serum glucose, TC, TG, and hepatic MDA levels (P < .05), whereas it increased serum HDL-C level and reactivated the hepatic antioxidant enzymes (P < .05) in alloxan-induced diabetic mice (P < .05). The hypoglycemic, hypolipidemic, and antioxidative activities of MG (100 mg/kg treatment) were all higher compared with all other diabetic groups and were similar to that observed for XiaoKeWan-pill (894 mg/kg; Guangzhou Zhongyi Pharmaceutical Co., Ltd., Guangzhou, China), a Chinese traditional antidiabetic drug. Antioxidant capacity evaluated in vitro showed that MG and mogroside V, which was the main component of MG, possessed strong oxygen free radical scavenging activities. These results demonstrate that the extract may have capacity to inhibiting hyperglycemia induced by diabetes, and the data suggest that administration of the extract may be helpful in the prevention of diabetic complications associated with oxidative stress and hyperlipidemia. We conclude that the extract should be evaluated as a candidate for future studies on diabetes mellitus.

Temporal Gene Expression Analysis of Human Coronary Artery Endothelial Cells Treated with Simvastatin

Increasing evidence indicates that the beneficial "pleiotropic" effects of statins on clinical events involve nonlipid mechanisms including the modification of blood vessel endothelial cell function. However, the involved molecular events and pathways are not completely understood. In the present study, Affymetrix microarrays were used to monitor the temporal gene expression of human coronary artery endothelial cells (HCAEC) treated with simvastatin (Sim) to gain insight into statins' direct effects on the endothelial function. We isolated and labeled mRNA from HCAEC treated with Sim for 0, 3, 6, 12, 24, and 48 h and hybridized these samples to Affymetrix GeneChip HG-U95Av2 to analyze the temporal gene expression profile. Out of 12,625 genes present on the HG-U95Av2 GeneChip, expression of 5,432 genes was detected. There were 1,475 of 5,432 genes that displayed the differential expression compared to baseline (0 h). Fifty-four genes were upregulated (< or = twofold) while 61 genes were downregulated ( > or = twofold) at 24-48 h after the Sim treatment. Many new target genes and pathways modulated by Sim were uncovered. This study indicates that many aspects of the pleiotropic effect of Sim on the endothelial cell function can be mediated by transcriptional control. Physiological function of 22% of 115 differentially expressed genes in Sim-treated HCAEC are currently unknown. These newly identified genes could be useful for new mechanistic study and new therapeutic modalities. Expressions of 13 out of 18 genes (> 70%) in the cell cycle/proliferation control process were significantly inhibited by the Sim treatment. CDC25B and ITGB4 gene expressions were validated by RT-PCR and Western blotting. Sim's inhibitory effect of on HCAEC growth was confirmed by the measurement of [3H]thymidine incorporation into the DNA synthesis. Further in-depth analysis of this effect may shed light on molecular mechanisms of Sim's beneficial inhibition of neointima formation in the atherosclerotic artery stenosis.

(5-Bromo-2-hydroxy-phen-yl)(phen-yl)methanone

In the title compound, C(13)H(9)BrO(2), the dihedral angle between the aromatic ring planes is 53.6 (1)°. The crystal structure is stabilized by intra-molecular O-H⋯O and inter-molecular C-H⋯O hydrogen bonding and C-H⋯π inter-actions.

N'-[(E)-1-(5-Bromo-2-hydroxy-phen-yl)ethyl-idene]benzohydrazide

The C=N double bond in the title compound, C(15)H(13)BrN(2)O(2), is transE configured and the dihedral angle between the aromatic ring planes is 22.3 (1)°. The crystal structure is stabilized by intra-molecular O-H⋯O and inter-molecular N-H⋯O hydrogen bonds.

Critical Role of PBEF Expression in Pulmonary Cell Inflammation and Permeability

Previous studies in our lab have identified pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury. This study continues to elucidate the underlying molecular mechanism of PBEF in the pathogenesis of acute lung injury in pulmonary cell culture models. Our results revealed that IL-1beta induced PBEF expression in pulmonary vascular endothelial cells at the transcriptional level and a -1535 T-variant in the human PBEF gene promoter significantly attenuated its binding to an IL-1beta-induced unknown transcription factor. This may underlie the reduced expression of PBEF and thus the lower susceptibility to acute lung injury in -1535T carriers. Furthermore, overexpression of PBEF significantly augmented IL-8 secretion and mRNA expression by more than 6-fold and 2-fold in A549 cells and HPAEC, respectively. It also significantly augmented IL-1beta-mediated cell permeability by 44% in A549 cells and 65% in endothelial cells. The knockdown of PBEF expression significantly inhibited IL-1beta-stimulated IL-8 secretion and mRNA level by 60% and 70%, respectively, and the knockdown of PBEF expression also significantly attenuated IL-1beta-induced cell permeability by 29% in epithelial cells and 24% in endothelial cells. PBEF expression also affected the expression of two other inflammatory cytokines (IL-16 and CCR3 genes). These results suggest that PBEF is critically involved in pulmonary vascular and epithelial inflammation and permeability, which are hallmark features in the pathogenesis of acute lung injury. This study lends further support to our finding that PBEF is a potential new target in acute lung injury.

Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor Via a Nonenzymatic and AP-1-dependent Mechanism

Although our previous studies found Pre-B-cell colony-enhancing factor (PBEF) as a highly up-regulated gene in acute lung injury that could stimulate expressions of other inflammatory cytokines, the underlying molecular mechanisms remain to be fully elucidated. Growing evidence indicates that PBEF is a nicotinamide phosphoribosyltransferase involved in the mammalian salvage pathway of NAD synthesis. This study was designed to determine whether the effect of PBEF to stimulate expressions of inflammatory cytokines depends on its enzymatic activity. We prepared two human PBEF mutant (H247E and H247A) recombinant proteins and overexpressing constructs for their overexpressions in A549 cells and confirmed that enzymatic activities of both mutants were nearly or completely abolished. Two mutants stimulated interleukin-8 (IL-8) expression at both the mRNA level and protein level just as equally effective as the wild-type PBEF did. These effects were due to the increased transcription, not the mRNA stability, of the IL-8 gene. Reporter gene assays and gel shift experiments indicated that AP-1 transcription factor is required to mediate these effects. SB203580, a p38 MAPK pathway inhibitor, and JNK inhibitor 1 can attenuate these effects. Both PBEF mutants similarly stimulated the expression of two other inflammatory cytokines: IL-16 and CCR3. These results indicate that PBEF stimulated expression of IL-8, IL-16, and CCR3 via its non-enzymatic activity. This effect is AP-1-dependent, in part via the p38 MAPK pathway and the JNK pathway. This finding reveals a new insight, which may manifest a novel role of PBEF in the pathogenesis of acute lung injury and other inflammatory disorders.

[Evaluation of Immunohistochemistry Staining and Cytologic Diagnosis by Using Cell Block Sections Prepared with Effusion Fluid Cytology Specimens]

To study the values of immunohistochemistry staining and cytological diagnosis by using cell block sections prepared with the effusion fluid cytology specimens.

Neuronal Protective Role of PBEF in a Mouse Model of Cerebral Ischemia

Pre-B-cell colony-enhancing factor (PBEF) (also known as nicotinamide phosphoribosyltransferase) is a rate-limiting enzyme in the salvage pathway for mammalian biosynthesis of nicotinamide adenine dinucleotide (NAD(+)). By synthesizing NAD(+), PBEF functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia is a major neural disorder with a high percentage of mortality and disability. Ischemia leads to energy depletion and eventually neuronal death and brain damage. This study investigated the role of PBEF in cerebral ischemia using a photothrombosis mouse model. Using immunostaining, we initially determined that PBEF is highly expressed in neurons, but not in glial cells in the mouse brain. To study the role of PBEF in ischemia in vivo, we used PBEF knockout heterozygous (Pbef+/-) mice. We showed that these mice have lower PBEF expression and NAD(+) level than do wild-type (WT) mice. When subjected to photothrombosis, Pbef+/- mice have significantly larger infarct volume than do age-matched WT mice at 24 hours after ischemia. Higher density of degenerating neurons was detected in the penumbra of Pbef+/- mice than in WT mice using Fluoro-Jade B staining. Our study shows that PBEF has a neuronal protective role in cerebral ischemia presumably through enhanced energy metabolism.

[A Comparison of Clinical Characteristics Between Radiological and Hemodynamic Massive Pulmonary Embolism]

To investigate the similarities and differences of massive pulmonary embolism (MPE) diagnosed by hemodynamic or radiological criteria, and therefore to improve physician's awareness of MPE from different aspects.

[Clinical Analysis of Hemodialysis Combined with Hemoperfusion for Treatment in Uremic Patients]

Nicotinamide Phosphoribosyltransferase in Human Diseases

Nicotinamide phosphoribosyltransferase (NAMPT) was first reported as a pre-B-cell colony enhancing factor in 1994 with little notice, but it has received increasing attention in recent years due to accumulating evidence indicating that NAMPT is a pleiotropic protein such as a growth factor, a cytokine, an enzyme and a visfatin. Now, NAMPT has been accepted as an official name of this protein. Because of NAMPT's multiple functions in a variety of physiological processes, their dysregulations have been implicated in the pathogenesis of a number of human diseases or conditions such as acute lung injury, aging, atherosclerosis, cancer, diabetes, rheumatoid arthritis and sepsis. This review will cover the current understanding of NAMPT's structure and functions with an emphasis on recent progress of nicotinamide phosphoribosyltransferase's pathological roles in various human diseases and conditions. Future directions on exploring its Terra incognita will be offered in the end.

[Effects of Receptor Interacting Protein 140 on the Biological Activity of Glia Cells]

To explore the effects of receptor interacting protein (RIP) 140 gene overexpression upon the in vitro proliferation, apoptosis, invasion and migration of microglioma cells.

RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin

The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.

A Multi-center Survey of Age of Sexual Debut and Sexual Behavior in Chinese Women: Suggestions for Optimal Age of Human Papillomavirus Vaccination in China

Cervical cancer is the second most common cancer among women worldwide, and over 85% of cervical cancers occur in developing countries such as China. Lack of resources for nationwide cervical cancer screening in China makes vaccination against oncogenic strains of HPV particularly important. Knowledge of age at sexual debut and sexual behavior is essential prior to implementation of a national vaccination program.

Impact of Genital Warts on Health Related Quality of Life in Men and Women in Mainland China: a Multicenter Hospital-based Cross-sectional Study

Information on the health-related quality of life (HRQoL) of patients with genital warts (GW) in populations in mainland China is still limited. The aim of the study was to use a generic instrument to measure the impact of genital warts on HRQoL in men and women in this setting.

A Multi-center Survey of HPV Knowledge and Attitudes Toward HPV Vaccination Among Women, Government Officials, and Medical Personnel in China

To assess knowledge of HPV and attitudes towards HPV vaccination among the general female population, government officials, and healthcare providers in China to assist the development of an effective national HPV vaccination program.

A Male with Chronic Thromboembolic Pulmonary Hypertension Caused by Isolated Noncompaction of Ventricular Myocardium

RNA-seq Analysis of Synovial Fibroblasts Brings New Insights into Rheumatoid Arthritis

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