FAM76B has been reported to be a nuclear speckle-localized protein with unknown function. In this study, FAM76B was first demonstrated to inhibit the NF-κB-mediated inflammatory pathway by affecting the translocation of hnRNPA2B1 in vitro. We further showed that FAM76B suppressed inflammation in vivo using a traumatic brain injury (TBI) mouse model. Lastly, FAM76B was shown to interact with hnRNPA2B1 in human tissues taken from patients with acute, organizing, and chronic TBI, and with different neurodegenerative diseases. The results suggested that FAM76B mediated neuroinflammation via influencing the translocation of hnRNPA2B1 in vivo during TBI repair and neurodegenerative diseases. In summary, we for the first time demonstrated the role of FAM76B in regulating inflammation and further showed that FAM76B could regulate the NF-κB-mediated inflammatory pathway by affecting hnRNPA2B1 translocation, which provides new information for studying the mechanism of inflammation regulation.

基于新型冠状病毒肺炎疫情影响和我国家庭体育发展的需求,以探讨疫情防控中我国家庭体育发展的形势契机、潜在价值、使命担当与发展路径,为取得疫情防控攻坚战的胜利和我国家庭体育的发展等提供参考与建言.研究认为:新型冠状病毒肺炎疫情唤醒了人们的生命健康意识、激发了体育参与热情、丰富了家庭体育环境和打通了体育协作壁垒是我国家庭体育发展的契机;维系生命安全、补充学-社体育、巩固全民健康和维护公共安全是我国家庭体育发展的价值;为生命安全保驾、为学校体育协作、为全民健康助力和为社会和谐护航是我国家庭体育发展的使命;树立正确的家庭体育意识观念、发挥政府的宣传与协调能力、保护潜在的家庭体育环境资源和制定长期的家庭体育防疫机制是我国家庭体育发展的路径.

Based on functional imaging methods of resting state DTI (Diffusion tensor imaging), this study reveals the structural changes and functional abnormalities of white matter fibers in patients with stroke from two levels of structure and function, and analyzes the effects of exercise on brain function remodeling in patients with stroke. The effects of the process, explore the central effect mechanism of scalp acupuncture to promote the recovery of motor function, and provide ideas for optimizing the rehabilitation treatment after stroke. There was no statistically significant difference in the general situation of the motion observation group and the conventional rehabilitation treatment group based on DTI technology at the time of enrollment, the NIHS S score, the fugl-meyerassessmenta score, the modified ashworth scale score, and the Brunnstrom staging assessment (P>0.05). After the week, 8 weeks, and 12 weeks of follow-up, the national institutes of health stroke scale, fugl-meyerassessmenta score, modified ashworth scale score, and Brunnstrom motor function staging score of the two groups of patients were significantly improved over time before treatment. The difference was statistically significant (P>0.05); the comparison between groups, the scores of patients in the action observation group and the difference before and after treatment were significantly higher than those in the conventional rehabilitation treatment group, and the difference was statistically significant (P>0.05). Conclusion: Action observation therapy can improve the degree of neurological deficits and promote the recovery of neurological function in patients with stroke. It can improve the score of motor function, reduce the muscle tension of key muscles in patients with stroke, and promote the recovery of motor function in patients.

Progranulin (PGRN) plays critical roles in inflammation, tumorigenesis, and neurodegeneration. PGRN levels in blood and cerebrospinal fluid (CSF) are being increasingly investigated as potential biomarkers for these disorders. However, the value of CSF PGRN as a biomarker has been limited because currently available commercial enzyme-linked immunosorbent assay (ELISA) kits have suboptimal sensitivity for detecting CSF PGRN. In this study, pairs of monoclonal antibodies (MAbs) were first screened from eleven monoclonal antiPGRN antibodies using indirect ELISA, then a sandwich ELISA was established using the 2 optimized MAbs. This system displayed high sensitivity, with a lower limit of detection of 60.0 pg/mL and a lower limit of quantification of 150 pg/mL. By using this ELISA system, we showed varied CSF PGRN levels in different brain disorders. For example, as compared with the normal controls, patients with Alzheimer disease or multiple sclerosis showed mildly increased CSF PGRN; those with aseptic encephalitis or neuropsychiatric systemic lupus erythematosus showed moderately increased CSF PGRN; those with bacterial leptomeningitis showed severely increased CSF PGRN. Additionally, determining CSF PGRN levels could monitor CNS metastasis and CSF seeding of carcinomas. These results indicate that this system can be valuable in studying the diagnostic and prognostic value of CSF PGRN in brain disorders.

DGKθ protein expression levels are closely related to the development of diseases including diabetes, cancer, and neuronal disease. To investigate the transcriptional regulation of the DGKθ gene, we used CRISPR/Cas9 to generate a DGKθ endogenous promoter luciferase reporter HepG2 cell line, in which the endogenous DGKθ promoter controls the expression of the luciferase reporter gene. To test the cell line, FXR, the transcription factor for upregulating the expression of DGKθ gene, was used to validate the cell line. Furthermore, the reported agonists for the expression of DGKθ, cAMP and GW4064, the known inhibitor for DGKθ enzyme activity, R59949, and a potential regulator for DGKθ enzyme expression, EGCG (the major catechin in green tea), were applied to the reporter cell line. The results indicated that these reagents could significantly regulate the expression of reporter luciferase. Finally, four transcription factors (E2F1, c-Myc, USF1, and Bmal1) potentially binding to the DGKθ gene’s upstream promoter region were tested. DGKθ expression was upregulated by c-Myc and downregulated by E2F1, which was also confirmed in wild-type HepG2 cells. We found that the cell line’s luciferase activity was directly correlated with DGKθ endogenous promoter activity, suggesting that it is liable and sensitive for studying DGKθ transcriptional regulation. The study provides a useful tool for high-throughput drug screening for the treatment of DGKθ-involved diseases.

分别了解末端释放训练和不同负荷强度的传统抗阻训练对高水平男子网球运动员平击发球速度与准确性的后激活增强效应及其最佳时间节点的变化.以7名高水平男子网球运动员为实验对象,采用4×6混合实验设计,利用手持式雷达测速仪与记录表分别对对照组、实验组的Pre与Post3、6、9、12、15 min时间节点上的平击发球速度与准确性进行科学验证与监测分析.结果发现:(1)对照组在12、15 min时间节点上的发球速度与Pre相比存在显著性差异(P＜0.05),在12 min节点上的发球准确性与Pre相比也存在显著性差异(P＜0.05);(2)末端释放训练组的发球速度在6 min节点达到了峰值,9 min后逐渐下降,3、6、9、12 min时间点均与Pre相比存在非常显著性差异(P＜0.01),15 min节点上的发球准确性达到了峰值,与Pre相比存在非常显著性差异(P＜0.01);(3)30％ 1RM传统抗阻组的发球速度在6 min节点达到最高值,之后保持平稳变化,在15 min时达最低点,6、9 min与Pre相比存在显著性差异(P＜0.05),6 min与3 min比较存在显著性差异(P＜0.05);6个时间节点上的发球准确性呈波浪式变化特征,在12 min时达到了峰值,其中,6 min节点的发球准确性与3 min相比存在非常显著性差异(P＜0.01),12 min与Pre相比也存在非常显著性差异(P＜0.01);(4)80％ 1RM传统抗阻组发球速度在3 min节点最低,然后缓慢上升,并在12 min时达到了峰值,9、12、15 min与Pro相比较存在非常显著性差异(P＜0.01),发球准确性在6 min达到峰值,与Pre相比存在非常显著性差异(P＜0.01);(5)在3 min和6 min节点上末端释放训练组发球速度与对照组相比存在非常显著性差异(P＜0.01),80％ 1RM传统抗阻组发球准确性在12 min节点与对照组比较存在显著性差异(P＜0.05).末端释放训练明显提高了高水平男子网球运动员3 min、6 min和9 min时间节点上的一发球速,其中在6min节点上达到了峰值,明显早于大负荷(80％ 1RM)传统抗阻训练在12 min和15 min的最佳PAP节点,末端释放训练和传统抗阻训练对网球平击发球准确性的PAP效应较小.

ObjectivesTo establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene.ResultsPCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line.ConclusionsWe successfully generated anovel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.

Heterozygous loss-of-function mutations in GRN, the progranulin gene, which result in progranulin (PGRN) protein haploinsufficiency, are a major cause of frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). PGRN is composed of seven and a half repeats of a highly conserved granulin motif that is cleaved to produce the granulin peptides A-G and paragranulin. To better understand the role of PGRN and granulin (Grn) peptides in the pathogenesis of neurodegeneration, we evaluated PGRN/Grn in brains of patients with Alzheimer disease, FTLD-TDP type A with or without GRN mutations, and normal individuals, using a panel of monoclonal antibodies against Grn peptides A-G. In the neocortex, Grn peptide-specific immunostains were observed, for example, membranous Grn E immunopositivity in pyramidal neurons, and Grn C immunopositivity in ramified microglia. In the hippocampus, Grn immunopositivity in the CA1 and CA2 regions showed disease-specific changes in both neurons and microglia. Most interestingly, in FTLD-TDP type A with GRN mutations, there is a 60% decrease in the density of Grn-positive microglia in the hippocampal CA1, suggesting that haploinsufficiency of the GRN mutations also extends to PGRN expression in microglia. This study provides important insights into future studies of the pathogenesis and treatment of FTLD-TDP.

Progranulin (PGRN), a highly glycosylated, secreted 593 amino acid precursor protein, is a multifunctional molecule that is critical for early embryogenesis, wound repair, inflammatory and tumorigenesis. PGRN can be proteolytically cleaved into seven cysteine-rich granulin (Grn) peptides: G, F, B, A, C, D and E. Both PGRN and its constituent Grn peptides have been implicated in a wide variety of biological activities. However, their functions are far from clear, and the lack of granulin domain-specific antibodies has hindered the progress of the functional study of PGRN and Grns. Monoclonal antibodies against GrnB, GrnA, GrnC and GrnF have been previously developed by our laboratory. In this study, we generated monoclonal antibodies (MAbs) against GrnD, GrnG and GrnE by using recombinant proteins HSA-GrnG, HSA-GrnD and HSA-GrnE as immunogens, and characterized them by indirect ELISA, Western blot and immunocytochemistry. Furthermore, the neutralizing activities of the MAbs against seven Grns were tested in vitro using the U251 cell line. This full antibody panel of MAbs against seven Grns will be a valuable tool for elucidating the biological roles of PGRN and Grns in different physiopathological processes, which will further promote the development of PGRN-based clinical diagnosis and therapy.

Human FAM76B (hFAM76B) is a 39 kDa protein that contains homopolymeric histidine tracts, a targeting signal for nuclear speckles. FAM76B is highly conserved among different species, suggesting that it may play an important physiological role in normal cellular functions. However, a lack of appropriate tools has hampered study of this potentially important protein. To facilitate research into the biological function(s) of FAM76B, murine monoclonal antibodies (MAbs) against hFAM76B were generated by using purified, prokaryotically expressed hFAM76B protein. Six strains of MAbs specific for hFAM76B were obtained and characterized. The specificity of MAbs was validated by using FAM76B-/- HEK 293 cell line. Double immunofluorescence followed by laser confocal microscopy confirmed the nuclear speckle localization of hFAM76B, and the specific domains recognized by different MAbs were further elucidated by Western blot. Due to the high conservation of protein sequences between mouse and human FAM76B, MAbs against hFAM76B were shown to react with mouse FAM76B (mFAM76B) specifically. Lastly, FAM76B was found to be expressed in the normal tissues of most human organs, though to different extents. The MAbs produced in this study should provide a useful tool for investigating the biological function(s) of FAM76B.

Human extracellular matrix protein-1 (hECM1), a secreted glycoprotein, is widely expressed in different tissues and organs. ECM1 has been implicated in multiple biological functions, which are potentially mediated by the interaction of different ECM1 domains with its ligands. However, the exact biological functions of ECM1 have not been elucidated yet, and the functional study of ECM1 has been partially hampered by the lack of sensitive and specific antibodies, especially those targeting different ECM1 domains. In this study, six strains of monoclonal antibody (MAb) against hECM1 were generated using purified, prokaryotically-expressed hECM1 as an immunogen. The MAbs were shown to be highly sensitive and specific, and suitable for western blot, immunoprecipitation assays and immunohistochemistry. Furthermore, the particular ECM1 domains recognized by different MAbs were identified. Lastly, the MAbs were found to have neutralizing activities, inhibiting the proliferation, migration and metastasis of MDA-MB-231 cells. In conclusion, the domain-specific anti-ECM1 MAbs produced in this study should provide a useful tool for investigating ECM1's biological functions, and cellular pathways in which it is involved.

研究以运动员左侧末端杠铃加速度、速度和高度等为指标对高丹抓举技术进行分析.采用2台SpeedCamMacroVis EoSens高速相机进行拍摄,通过SIMI-motion三维运动录像分析系统对其技术动作进行分析.研究结果:(1)成功时在A点杠铃加速度为0.39 m/s2,失败时为0.12 m/s2,说明成功时在杠铃离地瞬间用力较快,2次抓举A至D杠铃加速度和肩部移动距离的变化,说明在伸膝提铃阶段都有二次发力现象且上体过早后仰;(2)D至E杠铃加速度成功和失败时分别减少了0.07 m/s2和0.03 m/s2,说明在引膝阶段用力不连贯;(3)在F点成功时杠铃加速度最大为0.38 m/s2,失败时为0.41 m/s2,说明成功时用力节奏较差;(4)成功时左侧和右侧支撑点分别向后移动了0.17m和0.22 m,而失败时左侧和右侧支撑点分别向后移动了0.13m和0.11m,后跳之后两个支撑点没有在同一条水平线上,说明后跳之后两个支撑点发生前后偏离;(5)失败时杠铃的最高高度为1.04m,而成功时1.09m,失败时杠铃高度较低,在下蹲支撑阶段没有撑住杠铃导致其失败.把杠铃加速度的变化以折线图的形式来分析运动员技术动作,可以直观反映运动员的用力节奏,发现其技术动作的精细化程度不够,用力节奏较差,还需对其技术动作进行改进.

In glioblastomas, the surface glycoprotein CD133 (prominin-1) indicates the presence of cancer stem cells (CSCs), which are able to initiate tumor growth and are highly resistant to conventional chemo/radiotherapy. However, a number of studies have reported that certain CD133- glioma cells are able to self-renew and retain tumorigenic potential. In addition, the reliability of CD133 as a CSC marker is controversial due to inconsistent findings with regard to the prognostic values and distribution of CD133. Such controversies may be due to the detection limits using currently available anti-CD133 antibodies. In the present study, novel anti-human CD133 monoclonal antibodies (mAbs) were generated using two recombinant extracellular domains of human CD133: CD133 ectodomain 1 (amino acids 171-420) and CD133 ectodomain 2 (amino acids 507-716). One of the antibodies produced against CD133 ectodomain 2, C2E1, detected high expression levels of CD133 protein in glioblastoma U87 cells, in contrast to previous studies which did not detect CD133 expression in these cells. The cells exhibited a cytoplasmic distribution pattern of CD133 and produced a 95 kDa band following western blot analysis. In addition, C2E1 was able to bind the full-length glycosylated CD133 on the cell surface and inhibit the proliferation of tumor cells. Therefore, this antibody may be a valuable tool to study CD133 as a CSC marker and may be significant in future cancer treatments.

Background: One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.Results: We constructed a single adenoviral vector carrying in its E1 region a novel "all-in-one" Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.Conclusions: Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.

Current methods of treatment for lung carcinoma are ineffective for the majority of patients. Conditionally replicating adenoviruses (CRAds) represent a potential novel treatment for a number of neoplastic diseases, including lung carcinoma. The present study aimed to investigate the synergistic mechanisms underlying the anti-angiogenesis gene, arresten, and the apoptosis-inducing gene, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in order to evaluate their therapeutic potential in lung cancer. The two genes were expressed by CRAd, which was confirmed using reverse transcription-polymerase chain reaction and western blotting. In vitro analyses demonstrated that CRAd adenoviruses are capable of selectively inhibiting A549 lung cancer cell growth and replication but not in that of healthy cells. In vivo analyses demonstrated that the infection of A549 cell lines using CRAd armed with the two genes (CRAd-arresten-TRAIL) enhanced the tumor inhibition, compared with cells infected with CRAd-arresten, CRAd-TRAIL or CRAd, and with the control group. CRAd-arresten-TRAIL may therefore be useful in the treatment of lung cancer.

The progress of the application of adenovirus in cancer gene therapy is hindered by the lack of expression of native adenovirus receptor on a variety of cancer types. Hence, strategies are needed to retarget the adenoviral vector to non-native cellular surface receptors. In the present study, a new peptide SWDIAWPPLKVP, capable of selectively targeting a human glioblastoma cell line A172, was identified by direct biopanning of phage-display peptide libraries. The binding activity of the phage displaying SWDIAWPPLKVP peptide in A172 was more than 10-fold higher than that of the control phage. We then inserted the selected peptide SWDIAWPPLKVP into adenoviral hexon protein, and observed that the modified Ad5 had increased infectivity in A172 cells, compared with that in control cell lines. These findings demonstrated that a peptide acquired through phage display can mediate cell-specific Ad retargeting when inserted into Ad hexon, suggesting an approach for targeting adenoviral infection to specific cancer cells.

Zinc finger nuclease (ZFN) technology, which can be used to induce targeted genome correction in the presence of a DNA donor template, is becoming an attractive strategy for treating monogenic diseases. This strategy requires efficient delivery of ZFN and donor template into cells, ideally, in a single viral vector to achieve efficient genome editing and to avoid unwanted mutagenesis. In this study, we successfully produced a single adenoviral (Ad) vector with high titer that carried a ZFN expression cassette and a donor template simultaneously. We then demonstrated that this single Ad system could mediate efficient site-specific genome correction in vitro and ex vivo. The gene correction efficiency of the single Ad was significantly higher than that of the double Ad system. This novel vector will be a promising ZFN and donor delivery system for treatment of monogenic diseases.

目的:构建经嵌合纤维蛋白Ad5/35修饰的双表达hIL-12和hIFN-α2a的新型腺病毒载体并进行体外实验研究.方法:采用常规基因克隆和同源重组技术,构建在E3区携带hIL-12B(p40)和在纤维蛋白及E4区之间携带hIFN-α2a及eGFP表达框的经嵌合纤维蛋白Ad5/35修饰的嵌合型腺病毒骨架质粒载体pAd5/35-Backbone.E3-CMV-hIL-12B(p40)/E4-hIFN-α2a-eGFP.此外,采用分子克隆手段构建携带hIL-12A(p35)的腺病毒E1穿梭载体pAd5-E1-CMV-hIL-12A(p35).PCR法鉴定腺病毒骨架载体及穿梭载体质粒.将上述所获得的两种质粒载体经过PacI线性化后共转染HEK293细胞包装新型病毒载体.将纯化的腺病毒感染人恶性胶质瘤细胞U87MG,48小时后,荧光显微镜下观察报告基因eGFP的表达,RT-PCR检测hIL-12及hIFN-α2a目的基因的表达.结果:PCR法检测到所构建的腺病毒骨架载体及穿梭载体质粒成功携带所需的目的基因.成功包装并纯化获得新型腺病毒载体pAd5/35.E1-CMV-hIL-12A(p35)/E3-CMV-hIL-12B(p40)/E4-hIFN-α2a-eGFP.体外感染U87MG细胞,48h后荧光显微镜下可以观察到报告基因eGFP的表达,RT-PCR可以检测到hIL-12及hIFN-α2a目的基因的表达.结论:体外实验结果表明,成功制备的经嵌合纤维蛋白Ad5/35修饰的双表达hIL-12和hIFN-α2a新型腺病毒载体,为进一步探讨其在恶性脑胶质瘤动物模型中的体内治疗研究奠定了基础.

By used three-dimensional images method to analyze the final force lower limb motion of Yan Zekun and Hu Hailong who are the excellent young athletes in Shanxi province,seek different throwing kinematic parameters by three-dimensional motion analysis system,and find their features and shortcomings.This paper provides the theory evidence and reference for coaches.