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In JoVE (1)
Other Publications (13)
- Yi Chuan = Hereditas / Zhongguo Yi Chuan Xue Hui Bian Ji
- Pain
- Journal of Neurophysiology
- Journal of Manipulative and Physiological Therapeutics
- Planta
- Proceedings of the National Academy of Sciences of the United States of America
- Biochemical and Biophysical Research Communications
- Genome Biology
- Cell Research
- Current Opinion in Cell Biology
- Journal of Integrative Plant Biology
- Biophysical Journal
- Molecular Biotechnology
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Articles by Qiang Gan in JoVE
JoVE General
Хроматин иммунопреципитации (чип) использование Drosophila Ткани
Department of Biology, Johns Hopkins University
Недавно высокой пропускной последовательности технологий значительно увеличило чувствительность Хроматин иммунопреципитации (чип) эксперимент и побудило его применения с использованием очищенных клеток или расчлененные ткани. Здесь мы очертить метод использовать ChIP технику
Other articles by Qiang Gan on PubMed
[The New Advance of Coupled Transcription and Translation: Translation Within the Nuclei in Eukaryotes]
It is well known that the processes of transcription and translation are coupled in prokaryotes. However, in eukaryotes, shortly after the transcription of the primary transcript begins, modifications and processing occur. After the mature mRNA moleculars are transported from the nuclei to the cytoplasm, the translations begin. It shows that the processes of transcription and translation are not coupled in eukaryotes. But now Iborra et al localized translation sites with [3H] lysine or lysyl-tRNA tagged with biotin or BODIPY in mammalian cells and found that there exited coupled transcription and translation within the nuclei. They estimated that the nuclear translation accounted for about 10% to 15% of protein synthesis in the cell.
Thiamine, Pyridoxine, Cyanocobalamin and Their Combination Inhibit Thermal, but Not Mechanical Hyperalgesia in Rats with Primary Sensory Neuron Injury
Neuropathic pain after nerve injury is severe and intractable, and current drugs and nondrug therapies offer substantial pain relief to no more than half of affected patients. The present study investigated the analgesic roles of the B vitamins thiamine (B1), pyridoxine (B6) and cyanocobalamin (B12) in rats with neuropathic pain caused by spinal ganglia compression (CCD) or loose ligation of the sciatic nerve (CCI). Thermal hyperalgesia was determined by a significantly shortened latency of foot withdrawal to radiant heat, and mechanical hyperalgesia was determined by a significantly decreased threshold of foot withdrawal to von Frey filaments stimulation of the plantar surface of hindpaw. Results showed that (1) intraperitoneal injection of B1 (5, 10, 33 and 100 mg/kg), B6 (33 and 100 mg/kg) or B12 (0.5 and 2 mg/kg) significantly reduced thermal hyperalgesia; (2) the combination of B1, B6 and B12 synergistically inhibited thermal hyperalgesia; (3) repetitive administration of vitamin B complex (containing B1/B6/B12 33/33/0.5 mg/kg, for 1 and 2 wk) produced long-term inhibition of thermal hyperalgesia; and (4) B vitamins did not affect mechanical hyperalgesia or normal pain sensation, and exhibited similar effects on CCD and CCI induced-hyperalgesia. The present studies demonstrate effects of B vitamins on pain and hyperalgesia following primary sensory neurons injury, and suggest the possible clinical utility of B vitamins in the treatment of neuropathic painful conditions following injury, inflammation, degeneration or other disorders in the nervous systems in human beings.
CAMP and CGMP Contribute to Sensory Neuron Hyperexcitability and Hyperalgesia in Rats with Dorsal Root Ganglia Compression
Numerous studies have implicated the cAMP-protein kinase A (PKA) pathway in producing hyperexcitability of dorsal root ganglia (DRG) sensory neurons under conditions associated with pain. Evidence is presented for roles of both the cAMP-PKA and cGMP-protein kinase G (PKG) pathways in maintaining neuronal hyperexcitability and behavioral hyperalgesia in a neuropathic pain model: chronic compression of the DRG (CCD treatment). Lumbar DRGs were compressed by a steel rod inserted into the intervertebral foramen. Thermal hyperalgesia was revealed by shortened latencies of foot withdrawal to radiant heat. Intracellular recordings were obtained in vitro from lumbar ganglia after in vivo DRG compression. Activators of the cAMP-PKA pathway, 8-Br-cAMP and Sp-cAMPS, and of the cGMP-PKG pathway, 8-Br-cGMP and Sp-cGMPS, increased the hyperexcitability of DRG neurons already produced by CCD treatment, as shown by further decreases in action potential threshold and increased repetitive discharge during depolarization. The adenylate cyclase inhibitor, SQ22536, the PKA antagonist, Rp-cAMPS, the guanylate cyclase inhibitor, ODQ, and the PKG inhibitor, Rp-8-pCPT-cGMPS, reduced the hyperexcitability of CCD DRG neurons. In vivo application of PKA and PKG antagonists transiently depressed behavioral hyperalgesia induced by CCD treatment. Unexpectedly, application of these agonists and antagonists to ganglia of naïve, uninjured animals had little effect on electrophysiological properties of DRG neurons and no effect on foot withdrawal, suggesting that sensitizing actions of these pathways in the DRG are enabled by prior injury or stress. The only effect observed in uncompressed ganglia was modest depolarization of DRG neurons by PKA and PKG agonists. CCD treatment also depolarized DRG neurons, but CCD-induced depolarization was not affected by agonists or antagonists of these pathways.
Spinal Manipulation Reduces Pain and Hyperalgesia After Lumbar Intervertebral Foramen Inflammation in the Rat
To document potential mediating effects of the Activator-assisted spinal manipulative therapy (ASMT) on pain and hyperalgesia after acute intervertebral foramen (IVF) inflammation.
Functional Characterization of Rice OsDof12
DNA-binding with one finger (Dof) proteins are a large family of transcription factors involved in a variety of biological processes in plants. In rice, 30 different Dof genes have been identified through genome analysis. Here we report the functional characteristics of a rice Dof gene, OsDof12, which encodes a predicted Dof protein. The nuclear localization of OsDof12 was investigated by the transient expression assays of the OsDof12-GFP fusion protein in onion epidermal cells. Trans-activation assays in a yeast one-hybrid system indicated that OsDof12 had transcriptional activity. RNA expression analyses showed that the expression of OsDof12 was not tissue-specific in general and fluctuated at different development stages in rice. In addition, OsDof12 was strongly inhibited by dark treatments. The transgenic lines overexpressing OsDof12 showed early flowering under long-day (LD) conditions, whereas OsDof12 overexpression had no effect on flowering time under short-day (SD) conditions. In transgenic lines overexpressing OsDof12, the transcription levels of Hd3a and OsMADS14 were up-regulated under LD conditions but not SD conditions, whereas the expression of Hd1, OsMADS51, Ehd1 and OsGI did not change under LD and SD conditions. These results suggested that OsDof12 might regulate flowering by controlling the expression of Hd3a and OsMADS14.
A Transcriptomic Analysis of Superhybrid Rice LYP9 and Its Parents
By using a whole-genome oligonucleotide microarray, designed based on known and predicted indica rice genes, we investigated transcriptome profiles in developing leaves and panicles of superhybrid rice LYP9 and its parental cultivars 93-11 and PA64s. We detected 22,266 expressed genes out of 36,926 total genes set collectively from 7 tissues, including leaves at seedling and tillering stages, flag leaves at booting, heading, flowering, and filling stages, and panicles at filling stage. Clustering results showed that the F1 hybrid's expression profiles resembled those of its parental lines more than that which lies between the 2 parental lines. Out of the total gene set, 7,078 genes are shared by all sampled tissues and 3,926 genes (10.6% of the total gene set) are differentially expressed genes (DG). As we divided DG into those between the parents (DG(PP)) and between the hybrid and its parents (DG(HP)), the comparative results showed that genes in the categories of energy metabolism and transport are enriched in DG(HP) rather than in DG(PP). In addition, we correlated the concurrence of DG and yield-related quantitative trait loci, providing a potential group of heterosis-related genes.
Detection of Single Quantum Dots in Model Organisms with Sheet Illumination Microscopy
Single-molecule detection and tracking is important for observing biomolecule interactions in the microenvironment. Here we report selective plane illumination microscopy (SPIM) with single-molecule detection in living organisms, which enables fast imaging and single-molecule tracking and optical penetration beyond 300 microm. We detected single nanocrystals in Drosophila larvae and zebrafish embryo. We also report our first tracking of single quantum dots during zebrafish development, which displays a transition from flow to confined motion prior to the blastula stage. The new SPIM setup represents a new technique, which enables fast single-molecule imaging and tracking in living systems.
Monovalent and Unpoised Status of Most Genes in Undifferentiated Cell-enriched Drosophila Testis
Increasing evidence demonstrates that stem cells maintain their identities by a unique transcription network and chromatin structure. Opposing epigenetic modifications H3K27 me3 and H3K4 me3 have been proposed to label differentiation-associated genes in stem cells, progenitor and precursor cells. In addition, many differentiation-associated genes are maintained at a poised status by recruitment of the initiative RNA Polymerase II (Pol II) at their promoter regions, in preparation for lineage-specific expression upon differentiation. Previous studies have been performed using cultured mammalian embryonic stem cells. To a lesser extent, chromatin structure has been delineated in other model organisms, such as Drosophila, to open new avenues for genetic analyses.
Dynamic Regulation of Alternative Splicing and Chromatin Structure in Drosophila Gonads Revealed by RNA-seq
Both transcription and post-transcriptional processes, such as alternative splicing, play crucial roles in controlling developmental programs in metazoans. Recently emerged RNA-seq method has brought our understanding of eukaryotic transcriptomes to a new level, because it can resolve both gene expression level and alternative splicing events simultaneously. To gain a better understanding of cellular differentiation in gonads, we analyzed mRNA profiles from Drosophila testes and ovaries using RNA-seq. We identified a set of genes that have sex-specific isoforms in wild-type (WT) gonads, including several transcription factors. We found that differentiation of sperms from undifferentiated germ cells induced a dramatic downregulation of RNA splicing factors. Our data confirmed that RNA splicing events are significantly more frequent in the undifferentiated cell-enriched bag of marbles (bam) mutant testis, but downregulated upon differentiation in WT testis. Consistent with this, we showed that genes required for meiosis and terminal differentiation in WT testis were mainly regulated at the transcriptional level, but not by alternative splicing. Unexpectedly, we observed an increase in expression of all families of chromatin remodeling factors and histone modifying enzymes in the undifferentiated cell-enriched bam testis. More interestingly, chromatin regulators and histone modifying enzymes with opposite enzymatic activities are coenriched in undifferentiated cells in testis, suggesting that these cells may possess dynamic chromatin architecture. Finally, our data revealed many new features of the Drosophila gonadal transcriptomes, and will lead to a more comprehensive understanding of how differential gene expression and splicing regulate gametogenesis in Drosophila. Our data provided a foundation for the systematic study of gene expression and alternative splicing in many interesting areas of germ cell biology in Drosophila, such as the molecular basis for sexual dimorphism and the regulation of the proliferation vs terminal differentiation programs in germline stem cell lineages. The GEO accession number for the raw and analyzed RNA-seq data is GSE16960.
Epigenetic Regulation of Germ Cell Differentiation
Germ cells and somatic cells have the identical genome. However, unlike the mortal fate of somatic cells, germ cells have the unique ability to differentiate into gametes that retain totipotency and produce an entire organism upon fertilization. The processes by which germ cells differentiate into gametes, and those by which gametes become embryos, involve dramatic cellular differentiation accompanied by drastic changes in gene expression, which are tightly regulated by genetic circuitries as well as epigenetic mechanisms. Epigenetic regulation refers to heritable changes in gene expression that are not due to changes in primary DNA sequence. The past decade has witnessed an ever-increasing understanding of epigenetic regulation in many different cell types/tissues during embryonic development and adult homeostasis. In this review, we focus on recent discoveries of epigenetic regulation of germ cell differentiation in various metazoan model organisms, including worms, flies, and mammals.
Transcriptional Characteristics of Xa21-mediated Defense Responses in Rice
Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most destructive bacterial disease of rice. The cloned rice gene Xa21 confers resistance to a broad spectrum of Xoo races. To identify genes involved in Xa21-mediated immunity, a whole-genome oligonucleotide microarray of rice was used to profile the expression of rice genes between incompatible interactions and mock treatments at 0, 4, 8, 24, 72 and 120 h post inoculation (hpi) or between incompatible and compatible interactions at 4 hpi, respectively. A total of 441 differentially expressed genes, designated as XDGs (Xa21 mediated differentially expressed genes), were identified. Based on their functional annotations, the XDGs were assigned to 14 categories, including defense-related, signaling, transcriptional regulators. Most of the defense-related genes belonged to the pathogenesis-related gene family, which was induced dramatically at 72 and 120 hpi. Interestingly, most signaling and transcriptional regulator genes were downregulated at 4 and 8 hpi, suggesting that negative regulation of cellular signaling may play a role in the Xa21-mediated defense response. Comparison of expression profiles between Xa21- and other R gene-mediated defense systems revealed interesting common responses. Representative XDGs with supporting evidences were also discussed.
STED-SPIM: Stimulated Emission Depletion Improves Sheet Illumination Microscopy Resolution
We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 μm deep penetration into biological tissue.
Identification of Potential Antisense Transcripts in Rice Using Conventional Microarray
Natural antisense transcripts (NATs) are endogenous transcripts that contain reverse complementary sequences to other RNAs (usually called sense transcripts). NATs regulate the expression of sense transcripts in a wide range of species. The identification and analysis of NATs are the prerequisite to elucidate their functions. Microarray is a genome-wide method to detect gene expression. However, conventional microarrays do not contain the specific probes of NATs; thus, they cannot be utilized to detect NATs. In this article, we developed a novel method to identify potential NATs with the conventional microarrays. In this method of our study, we labeled the first strand cDNA from one sample with Cy5 and labeled the second strand cDNA from another sample with Cy3, and then hybridized these labeled samples with oligonucleotide microarray. Using this method, we identified 920 potential NATs in rice variety Nipponbare. Among these potential NATs, 88 of them were confirmed by either full-length cDNA or orientated ESTs (expressed sequence tags). This is the first time that a conventional oligonucleotide microarray was employed to identify NATs in rice.
