Abstract:The typeⅡclustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 system has been used as a gene-targeting technology as ZFN and TALEN to meditate multiple genome editing. Unlike ZFN and TALEN directly binding to the specific DNA sequence to generate a double-strands break, the engineered CRISPR/Cas9 system has been demonstrated that Cas9 nuclease was directed by short RNAs to induce site-specific cleavage in complex genome. The advantage of CRPSR/Cas9 system is easy to be constructed and low cost compared with ZFN and TALEN. So, it is considered that it will replace existing technique. Here we review the CRISPR/Cas development history, classification, mechanism, progress and application of this new genome editing technology. This review will provide a useful reference for researchers who are interested in applying this new technique in their studies.

Abstract:To solve the shortage of blood sources has become an urgent need in the current medical. One of the important solutions is functional production of erythrocytes in vitro using stem/progenitor cells. But enucleation remains one of the critical rate-limiting steps. Even though reticulocytes could be produced in vitro, the low efficiency should be concerned. To uncover the molecular mechanism of erythroblast enucleation will facilitate ex vivo production of functional erythrocytes. This review summarizes three prevalent enucleation mechanisms, including apoptosis, asymmetric cytokinesis and vesicle trafficking, discusses proteins and microRNAs playing important role in the process, and evaluates the prospects for ex vivo production of red blood cells.

Abstract:Approximately 45% of the human genome is occupied by transposable elements. Transposable elements have had an important impact on the structure and function of human genomes. Some of these elements are still capable of transposing, and their movements often cause diseases. Long interspersed nuclear element 1 (LINE-1) is the only active autonomous transposon in humans, and it mediates the mobilization of nonautonomous elements. Recently, great progress has been made in understanding the biology of LINE-1. Here, we briefly review the structure of LINE-1, the mechanism of its mobilization, and its impact on human genome and human health. We also highlight the mechanisms that host cell has evolved to control LINE-1 replication. Given the similarity of the life cycle between LINE-1 and retroviruses, the LINE-1 research may also shed new light on the biology of retroviruses.

Abstract:Exosomes are nano-sized vesicles ranging from 40 to 100 nm released from various cell types under different functional conditions. They originate from the endosomal system and are secreted by cells upon fusion of multivesicular endosomes with the plasma membrane. Exosomes were initially regarded as clearing mechanisms that discard unwanted proteins and membranes of reticulocytes during their maturation. However, they are believed to play much wider ranges of roles recently other than clearing, especially information conduction among different cell types. Exosomes deliver bioactive molecules including proteins and RNAs to the target cells by interacting with cell surface receptors or fusing with plasma membrane and release cargo RNAs to target cells, they can also release functionally active molecules in the vicinity of the recipient cells. Recent research focused on their roles in the development and progression of tumors and nervous system diseases, on the other hand, they are also promising biomarkers for clinical diagnostic purpose and ideal therapeutic tools for biodelivery.

Abstract:无

Abstract:Regulation of inflammatory cytokines is a critical stage in inflammation, an important factor in autoimmune disease and cancer. Nitric oxide (NO) is known to be an important regulator of inflammatory cytokines, however, most existing studies focus on the role of NO synthesis in the regulation of inflammatory cytokines, and little is known about the role of NO metabolism. Since S-nitrosoglutathione reductase (GSNOR) is a key protein in the control of NO metabolism, investigating its role in inflammation will be important for understanding the role of NO metabolism. Here we found that GSNOR transcription and protein expression is downregulated by lipopolysaccharide (LPS) in RAW264.7 cells, an inflammatory cell model. Inhibitors of MEK1/2, p38 and PI3K significantly attenuate the decrease in GSNOR transcription. Furthermore, inhibition of the enzyme activity of GSNOR promoted expression of LPS-induced inflammatory cytokines IL-1β, IL-6 and TNF-α, whereas overexpression of GSNOR had the opposite effect. The anti-inflammatory drug trichostatin A (TSA) rescued the downregulation of GSNOR expression by LPS. Furthermore, inhibition of GSNOR impaired the anti-inflammatory effect of TSA by increasing the expression of IL-6 and TNF-α. In conclusion, our work reveals a new mechanism used by macrophage cells to enhance the inflammatory response by simultaneously upregulating inducible nitric oxide synthase(iNOS) and downregulating GSNOR, thus expanding our understanding of NO metabolism in inflammatory responses. This study shows that GSNOR is a novel regulator of inflammation and may be a potential target for the regulation of NO-mediated inflammation.

Abstract:Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin. The plasticity and dynamics of higher-order chromatin fiber have been widely thought as the key regulators of transcription and other biological processes inherent to DNA. Elucidating how nucleosomal arrays can be folded into higher-order chromatin fibers is essential to understand the dynamics of chromatin structure. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, which comprises 147 base pairs of DNA wrapped in 1.7 superhelical turns around an octamer of histones, has been solved at the atomic resolution, there is still much controversy over the chromatin structure at the higher-order level. Here, we built an in vitro chromatin reconstitution system which adopts histone octamers and arrays of 177 bp and 200 bp repeat of the Widom 601 DNA sequence. Taking advantage of this system, we have obtained highly regular spaced and soluble nucleosome arrays, and folded the arrays into 30 nm chromatin fibers with the existence of linker histone H1 or MgCl₂ respectively. Several electron microscopic techniques, including metal shadowing, negative staining and Cryo-EM, have been used to investigate the morphology of the reconstituted 30 nm chromatin fibers. Our results suggest that both histone H1 and divalent Mg²⁺ can help the formation of 30 nm chromatin fibers, but the resulted chromatin fibers display different topologically architectures. To investigate how the length of linker histone may affect the architecture of chromatin，we measured the diameters of the reconstituted 30 nm chromatin fibers with different nucleosome repeat lengths (NRLs) of 177 and 200 bp and found that these two classes of chromatin fibers present different diameters (P < 0.05).

Abstract:Compound YSY-01A is a recently synthesized proteasome inhibitor. It has been proved for potent growth-inhibitory effect on tumor cells in previous studies. However, the effect of YSY-01A on tumor angiogenesis remains unclear. Our research aims to reveal the inhibition effect and mechanism of YSY-01A on tumor-induced angiogenesis. Firstly, we combined the Sulforhodamine B (SRB) assay and Transwell co-culture model to observe the inhibition of YSY-01A on human umbilical vein endothelial cells (HUVECs) proliferation induced by tumor cells (HT-29 cells). In succession, high content screening (HCS) assay was used to investigate effect of YSY-01A on NF-κB nuclear translocation in HT-29 cells. Finally, Western blot was used to measure the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in HT-29 cells inhibited by YSY-01A. To further determine mechanism of inhibition, SRB and HCS methods were used to investigate the effect of YSY-01A against HUVECs proliferation and motility, respectively. The results showed that YSY-01A could prohibit HUVECs proliferation induced by HT-29 cells in a concentration-dependent manner. Furthermore, YSY-01A significantly inhibited NF-κB nuclear translocation and reduced the expression of HIF-1α and VEGF in HT-29 cells. Further investigation revealed concentration-dependent suppress of YSY-01A on HUVECs proliferation and motility without obvious cytotoxic effect. In conclusion, through proteasome inhibition, YSY-01A could down-regulate pro-angiogenesis factors expression in tumor cells and exhibit remarkable anti-angiogenesis activity on vascular endothelial cells.

Abstract:Improvement of blood flow and promotion of neovascularization are important therapeutic measures for ischemic peripheral vascular diseases. Since apolipoproteinA(ApoA) is a glycoprotein with repetitive kringle domains exhibiting 75% to 98% structural homology with plasminogen(Plg), ApoA may also have a negative effect on endothelial progenitor cell (EPC)-induced vasculogenesis through Plg-like inhibitory effects on EPC proliferation, adhesion, migration and vasculogenesis. To evaluate the effect of ApoA on EPCs-induced vasculogenesis. ApoA was stably expressed and prepared from COS-7 cell line which were transfected with the expression plasmids pSG-5 encoding human ApoA cDNA，then purified by immunoaffinity chromatography; EPCs were isolated from the bone marrow of ApoA transgenic mice, wild-type litter mates and normal mice. These cells were cultured without or with ApoA before transplantation. Hindlimb ischemia models were surgically induced in mice, which then received an intravenously injection of 3×10⁵ EPCs. At 3, 7 and 14 days post EPC transplantation，the adhesion, migration abilities and capillary density in calf muscles were assessed. Results indicate that ApoA significantly reduced the adhesion and migration abilities of EPCs. Furthermore, the tubule-like formation of EPCs on Matrigel gels was damaged. In vivo experiments showed the number of EPCs home to ischemic peripheral vascular, and the number of capillary vessels decreased significantly in ApoA transgenic mice. This study demonstrated that ApoA could attenuate the adhesion, migration, and homing abilities of EPCs and could impair the vasculogenesis ability of EPCs.

Abstract:Ryanodine receptor (RyR) locating in the sarcoplasmic reticulum(SR) is one of the most important calcium release channels. It is known that RyR is rich in functional thiol groups, which play crucial roles in controlling channel gating, therefore the protein is quite sensitive to the redox environment. The main purpose of this research is to understand how the RyR1 modulators in general affect the channel gating according to their electron transfer properties. In this research, methods such as CPM labeling, photon correlation spectroscopy (PCS) and [³H]-ryanodine binding experiments were used to detect the change of free thiols' population, the diameter and distribution of protein complex and the gating state of RyR1. Furthermore, we used photon bleaching assay to determine the electron transfer property of the modulators. The results show that the channel activators and thiol oxidants appear to be electron acceptors that affect RyR1 in the same manner, which is to decrease the free thiols on RyR1 and to increase the size of the protein crosslinking clusters. In contrary, channel inhibitors and thiol reductants appear to be electron donors and their effects on RyR1 are opposite to activators and oxidants. Therefore, we propose that the similar electron transfer properties of channel modulators results in the similar effects on RyR1 by changing the local redox environment around the active thiols, inducing changes in thiol/disulfide balance and RyR1 gating.

Abstract:Glutamate excitotoxicity of cerebellar granule neurons (CGN) is often used as a model for studying the pathogenesis of acute, chronic neurodegenerative diseases and inflammation-induced neurological diseases. However, reports of culture methods for CGN and conditions for their stimulation vary considerably, making it difficult to compare reports from different laboratories. Here, we compare different methods and optimize the model as follows: Culture plates were coated overnight at room temperature with Poly-L-lysine at a concentration of over 50 mg/L. Cerebellum tissues were then shredded and digested with trypsin (0.025%) for 15 min at 37℃ with shaking in the presence of DNaseⅠ to eliminate DNA released from broken cells. In order to remove impurities and cell debris, samples were centrifuged respectively after tissue shredding, digestion and mechanical dispersion. Samples were then homogenized by pipetting and allowed to settle three times to increase the yield of CGN. Best results were achieved when settling time was extended to 15 min before collecting the supernatant cells. Conditional BME was used as the culture medium since our data showed that CGNs are more sensitive to glutamate in conditioned than in fresh BME, neurobasal medium or Locke's buffer. Cells cultured for 9 DIV (days in vitro) were treated with 100 μmol/L glutamate to generate moderate excitotoxicity. The model was validated by examining intracellular calcium dynamics and c-fos expression. Results show that this method is stable and reproducible and may be helpful for researchers using the excitotoxicity model to study neuropathological processes.