Animals ; Antigens, Surface/genetics* ; Cell Communication/genetics* ; Copulation/physiology* ; Fallopian Tubes/metabolism* ; Female ; Fertilization/genetics* ; GPI-Linked Proteins/genetics* ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/metabolism* ; Litter Size ; Luminescent Proteins/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism* ; Reproduction/genetics* ; Signal Transduction ; Sperm Count ; Sperm Motility/genetics* ; Spermatozoa/metabolism* ; Uterus/metabolism*

Although extensively studied, the exact role of sleep in learning and memory is still not very clear. Sleep deprivation has been most frequently used to explore the effects of sleep on learning and memory, but the results from such studies are inevitably complicated by concurrent stress and distress. Furthermore, it is not clear whether there is a strict time-window between sleep and memory consolidation. In the present study we were able to induce time-locked slow-wave sleep (SWS) in mice by optogenetically stimulating GABAergic neurons in the parafacial zone (PZ), providing a direct approach to analyze the influences of SWS on learning and memory with precise time-windows. We found that SWS induced by light for 30 min immediately or 15 min after the training phase of the object-in-place task significantly prolonged the memory from 30 min to 6 h. However, induction of SWS 30 min after the training phase did not improve memory, suggesting a critical time-window between the induction of a brief episode of SWS and learning for memory consolidation. Application of a gentle touch to the mice during light stimulation to prevent SWS induction also failed to improve memory, indicating the specific role of SWS, but not the activation of PZ GABAergic neurons itself, in memory consolidation. Similar influences of light-induced SWS on memory consolidation also occurred for Y-maze spatial memory and contextual fear memory, but not for cued fear memory. SWS induction immediately before the test phase had no effect on memory performance, indicating that SWS does not affect memory retrieval. Thus, by induction of a brief-episode SWS we have revealed a critical time window for the consolidation of hippocampus-dependent memory.
Animals ; Cues ; Electroencephalography ; Electromyography ; Evoked Potentials, Motor ; physiology ; Fear ; psychology ; Glutamate Decarboxylase ; metabolism ; Hippocampus ; physiology ; Light ; Luminescent Proteins ; genetics ; metabolism ; Maze Learning ; physiology ; Memory Consolidation ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Sleep Deprivation ; Sleep, Slow-Wave ; physiology ; Time Factors ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

Drosophila dEAAT2, a member of the excitatory amino-acid transporter (EAAT) family, has been described as mediating the high-affinity transport of taurine, which is a free amino-acid abundant in both insects and mammals. However, the role of taurine and its transporter in hearing is not clear. Here, we report that dEAAT2 is required for the larval startle response to sound stimuli. dEAAT2 was found to be enriched in the distal region of chordotonal neurons where sound transduction occurs. The Ca imaging and electrophysiological results showed that disrupted dEAAT2 expression significantly reduced the response of chordotonal neurons to sound. More importantly, expressing dEAAT2 in the chordotonal neurons rescued these mutant phenotypes. Taken together, these findings indicate a critical role for Drosophila dEAAT2 in sound transduction by chordotonal neurons.
Acoustic Stimulation ; Action Potentials ; genetics ; Animals ; Animals, Genetically Modified ; Auditory Pathways ; physiology ; Calcium ; metabolism ; Drosophila ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Excitatory Amino Acid Transporter 2 ; genetics ; metabolism ; Hearing ; genetics ; Larva ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; genetics ; Nervous System ; cytology ; Neurons ; metabolism

The brain has very high energy requirements and consumes 20% of the oxygen and 25% of the glucose in the human body. Therefore, the molecular mechanism underlying how the brain metabolizes substances to support neural activity is a fundamental issue for neuroscience studies. A well-known model in the brain, the astrocyte-neuron lactate shuttle, postulates that glucose uptake and glycolytic activity are enhanced in astrocytes upon neuronal activation and that astrocytes transport lactate into neurons to fulfill their energy requirements. Current evidence for this hypothesis has yet to reach a clear consensus, and new concepts beyond the shuttle hypothesis are emerging. The discrepancy is largely attributed to the lack of a critical method for real-time monitoring of metabolic dynamics at cellular resolution. Recent advances in fluorescent protein-based sensors allow the generation of a sensitive, specific, real-time readout of subcellular metabolites and fill the current technological gap. Here, we summarize the development of genetically encoded metabolite sensors and their applications in assessing cell metabolism in living cells and in vivo, and we believe that these tools will help to address the issue of elucidating neural energy metabolism.
Animals ; Biosensing Techniques ; Brain ; cytology ; metabolism ; Cytological Techniques ; Energy Metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Time Factors

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2) neurons. CCL2 increased NMDA-induced currents in CCR2/VGLUT2 neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.
Animals ; Benzoxazines ; pharmacology ; therapeutic use ; Chemokine CCL2 ; antagonists & inhibitors ; genetics ; metabolism ; pharmacology ; Excitatory Amino Acid Agents ; pharmacology ; Excitatory Amino Acid Agonists ; pharmacology ; Female ; Freund's Adjuvant ; toxicity ; Hyperalgesia ; chemically induced ; metabolism ; prevention & control ; Long-Term Potentiation ; drug effects ; physiology ; Luminescent Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myelitis ; chemically induced ; drug therapy ; metabolism ; Neurons ; drug effects ; Pain Management ; Somatostatin ; genetics ; metabolism ; Spinal Cord ; cytology ; Spiro Compounds ; pharmacology ; therapeutic use ; Vesicular Glutamate Transport Protein 2 ; genetics ; metabolism ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

OBJECTIVETo construct a eukaryotic expression vector to express the hepatitis E virus protein open reading frame 3 (ORF3) and investigate the intracellular location of the expressed protein using the baby hamster kidney (BHK-21) fibroblast cell line.

METHODSThe ORF3 gene was amplified by RT-PCR, cloned into the HindIII and EcoRI sites in the multicloning site of the pDsRed-Monomer-N1mammalian expression vector that encodes a red fluorescent protein (DsRed), and confirmed by restriction enzyme digestion and sequencing. The recombinant plasmid was then transfected into BHK-21 cells via the Lipofectamine 2000 reagent; the subsequent ORF3 gene overexpression was confirmed by RT-PCR and the protein expression and location was detected by Western blotting and immunofluorescence assay.Results TThe pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully constructed. After transfection into BHK-21 ceils, the ORF3 gene was transcribed and expressed, and the ORF3 protein was mainly located in the cytoplasm, where it could react with a specific antibody.

CONCLUSIONThe ORF3-DsRed fusion protein was mainly located in the cytoplasm of BHK-21 fibroblasts, and may represent a useful tool for research on the role of this protein in HEV infection.

Animals ; Cell Line ; Cricetinae ; Cytoplasm ; Fibroblasts ; metabolism ; Genetic Vectors ; genetics ; Hepatitis E virus ; metabolism ; Luminescent Proteins ; Open Reading Frames ; Plasmids ; Recombinant Proteins ; Transfection ; Viral Proteins ; metabolism

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r2 = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r2 = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.
Animals ; Blotting, Western ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Immunohistochemistry ; Luciferases, Firefly/genetics ; Luminescent Measurements ; Mice ; Mice, Nude ; Neoplasm Staging ; *Signal Transduction ; TOR Serine-Threonine Kinases/*metabolism ; Transplantation, Heterologous ; Urinary Bladder Neoplasms/*metabolism/pathology/veterinary

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
Animals ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; transplantation ; Gene Knock-In Techniques ; Genes, Reporter ; Homologous Recombination ; Humans ; Luminescent Proteins ; genetics ; Mice ; Mice, SCID ; Nestin ; genetics ; Neural Stem Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Teratoma ; pathology

Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.
Amino Acid Sequence ; Base Sequence ; genetics ; Cloning, Molecular ; DNA Primers ; biosynthesis ; Gene Expression Regulation, Fungal ; Genetic Vectors ; Luminescent Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Synthetic Biology ; Transformation, Genetic