BACKGROUND The DYNC1H1 gene encodes a part of the dynamic protein,and the protein mutations may further affect the growth and development of neurons,resulting in degeneration of anterior horn cells of the spinal cord,...BACKGROUND The DYNC1H1 gene encodes a part of the dynamic protein,and the protein mutations may further affect the growth and development of neurons,resulting in degeneration of anterior horn cells of the spinal cord,and a variety of clinical phenotypes finally resulting in axonal Charcot-Marie-Tooth disease type 20(CMT20),mental retardation 13(MRD13)and spinal muscular atrophy with lower extremity predominant 1(SMA-LED).The incidence of the disease is low,and it is difficult to diagnose,especially in children.Here,we report a case of DYNC1H1 gene mutation and review the related literature to improve the pediatrician’s understanding of DYNC1H1 gene-related disease to make an early correct diagnosis and provide better services for children.CASE SUMMARY A 4-mo-old Chinese female child with adducted thumbs,high arch feet,and epileptic seizure presented slow response,delayed development,and low limb muscle strength.Electroencephalogram showed abnormal waves,a large number of multifocal sharp waves,sharp slow waves,and multiple spasms with a series of attacks.High-throughput sequencing and Sanger sequencing identified a heterozygous mutation,c.5885 G>A(p.R1962H),in the DYNC1H1 gene(NM 001376)of the proband,which was not identified in her parents.Combined with the clinical manifestations and pedigree of this family,this mutation is likely pathogenic based on the American Academy of Medical Genetics and Genomics guidelines.The child was followed when she was 1 year and 2 mo old.The magnetic resonance imaging result was consistent with the findings of white matter myelinated dysplasia and congenital giant gyrus.The extensive neurogenic damage to the extremities was considered,as the results of electromyography showed that the motor conduction velocity and sensory conduction of the nerves of the extremities were not abnormal,and the degree of fit of the children with severe contraction was poor.At present,the child is 80 cm in length and 9 kg in weight,with slender limbs and low muscle strength,and still does not raise her head.She cannot sit or speak.Speech,motor,and mental development was significantly delayed.There is still no effective treatment for this disease.CONCLUSION We herein report a de novo variant of DYNC1H1 gene,c.5885 G>A(p.R1962H),leading to overlapping phenotypes(seizure,general growth retardation,and muscle weakness)of CMT20,MRD13,and SMA-LED,but there is no effective treatment for such condition.Our case enriches the DYNC1H1 gene mutation spectrum and provides an important basis for clinical diagnosis and treatment and genetic counseling.展开更多
[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the s...[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.展开更多
Age-related macular degeneration(AMD) is the leading cause of irreversible blindness in the developed world. The quality of life of both patients and families is impacted by this prevalent disease. Previously, macular...Age-related macular degeneration(AMD) is the leading cause of irreversible blindness in the developed world. The quality of life of both patients and families is impacted by this prevalent disease. Previously, macular degeneration had no known effective treatment. Today, vitamins for non-exudative AMD and intravitreal injection of medications for its exudative form are primary forms of current treatment. Modern advances in molecular science give rise to new possibilities of disease management. In the year 2003 the sequencing of the entire human genome was completed. Since that time, genes such as complement factor H, high-temperature requirement factor A1, and age-relateed maculopathy susceptibility 2 have been discovered and associated with a higher risk of AMD. A patient's genetic make-up may dictate the effectiveness of current or future therapeutic options. In addition, utilizing genetic data and incorporating it into new treatments(such as viral vectors) may lead to longer-lasting(or permanent) VEGF blockade and specific targeting of complement related genes. There have also been considerable advances in stem cell directed treatment of AMD. Retinal pigment epithelial(RPE) cells can be derived from human embryonic stem cells, induced pluripotent stem cells, or adult human RPE stem cells. Utilizing animal models of RPE and retinal degeneration, stem cell-derived RPE cells have been successfully implanted into the subretinal space. They have been injected as a cell mass or as a pre-prepared monolayer on a thin membrane. Visual recovery has been demonstrated in a retinal dystrophic rat model. Preliminary data on 2 human subjects also demonstrates possible early visual benefit from transplantation of stem cell-derived RPE. As more data is published, and as differentiation and implantation techniques are optimized, the stabilization and possible improvement of vision in individuals with non-exudative macular becomes a real possibility. We conclude that the technologic advances that continue to unfold in both genetic and stem cell research offer optimism in the future treatment of AMD.展开更多
目的总结1个以下肢受累为主的脊髓性肌萎缩症(spinal muscular atrophy with lower extremity predominant,SMALED)家系的临床、电生理及影像特点,并对该家系进行致病突变分析。方法收集2020年8月首都医科大学宣武医院确诊为常染色体显...目的总结1个以下肢受累为主的脊髓性肌萎缩症(spinal muscular atrophy with lower extremity predominant,SMALED)家系的临床、电生理及影像特点,并对该家系进行致病突变分析。方法收集2020年8月首都医科大学宣武医院确诊为常染色体显性遗传的1例SMALED患者的家系资料。采集先证者及其女儿的临床资料,进行血常规、肝功能、肌酸激酶等检验,肌电图和神经传导检查,脊髓、头颅、双下肢MRI检查,表型匹配分析,对先证者进行SMN1基因拷贝数检测和全外显子组测序分析。按照美国医学遗传学与基因组学学会(American College of Medical Genetics and Ge-nomics,ACMG)和美国分子病理学会(Association for Molecular Pathology,AMP)基因变异解读标准和指南进行致病性分析,对先证者及其女儿进行Sanger验证。结果家系调查发现,先证者及其女儿表型与SMALED表型匹配。两人均存在DYNC1H1基因(c.1792C>T;p.R598C)杂合突变,根据ACMG和AMP指南考虑为强致病性突变。结论在2例患者中鉴定出DYNC1H1基因p.R598C的致病性突变,符合常染色体显性遗传方式,提示对中国人群SMALED的研究需进行DYNC1H1基因检测。展开更多
Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping gen...Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.展开更多
The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evoluti...The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.展开更多
The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analys...The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.展开更多
Objective To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. Methods HeLa epithelial cells were treated with either histone...Objective To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. Methods HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls. Results Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells. Conclusion SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.展开更多
H5N1 influenza represents one of the great challenges to public health.Some H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to t...H5N1 influenza represents one of the great challenges to public health.Some H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to their natural hosts.Here,we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus.We demonstrated that,compared to the GS/65 virus,the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways(i.e.,RIG-I and MDA5) in ducks.We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes,while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus.We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis(WGCNA).Moreover,we first demonstrated that the duck BCL2 L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently.These analyses,in combination with our comprehensive transcriptomic data,provided global view of the molecular architecture for the interaction between host and H5N1 viruses.展开更多
Objective: The nm23 gene is one of the tumor metastatic suppressor genes. The expression of nm23H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including...Objective: The nm23 gene is one of the tumor metastatic suppressor genes. The expression of nm23H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including breast, colorectal, gastric, hepatocellular and gallbladder carcinomas. In this study, the immunohistochemical staining of nm23H1 protein in human nasopharyngeal carcinoma (NPC) was examined, and the relationship between nm23H1 and both metastasis and prognosis of patients with NPC was also investigated. Methods: Routine LSAB immunohistochemistry with the nm23H1 monoclonal murine antibody was employed to study the expression of nm23H1 protein in 95 paraffinembedded specimens of NPC treated at our hospital. The clinical pathologic data and results of followup were also retrieved. Comparisons between patients with and without expression of nm23H1 protein with respect to metastasis, locoregional recurrence and survival were performed using Log rank test. Multivariate prognostic analyses were performed by using Cox's regression model. Results: Nm23H1 negative expressive tumors were associated with a higher incidence of lymphnode metastasis (86.7%) than those of nm23H1 positive (48.6%, P<0.01). Nm23H1 negative expressive tumors were associated with a high incidence of recurrence and distant metastasis after radiotherapy (P<0.05). A significant association was found between expression of nm23H1 and prognosis (P<0.01). The expression of nm23H1 indicated favorable prognosis. Conclusion: It was suggested that nm23 H1 negative expression was significantly associated with lymphnode metastasis, recurrence and distant metastasis. Nm23H1 may have value for predicting the prognosis of NPC.展开更多
To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defectiv...To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.展开更多
In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of str...In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.展开更多
基金Supported by Jinan Science and Technology Project,No.201805014。
文摘BACKGROUND The DYNC1H1 gene encodes a part of the dynamic protein,and the protein mutations may further affect the growth and development of neurons,resulting in degeneration of anterior horn cells of the spinal cord,and a variety of clinical phenotypes finally resulting in axonal Charcot-Marie-Tooth disease type 20(CMT20),mental retardation 13(MRD13)and spinal muscular atrophy with lower extremity predominant 1(SMA-LED).The incidence of the disease is low,and it is difficult to diagnose,especially in children.Here,we report a case of DYNC1H1 gene mutation and review the related literature to improve the pediatrician’s understanding of DYNC1H1 gene-related disease to make an early correct diagnosis and provide better services for children.CASE SUMMARY A 4-mo-old Chinese female child with adducted thumbs,high arch feet,and epileptic seizure presented slow response,delayed development,and low limb muscle strength.Electroencephalogram showed abnormal waves,a large number of multifocal sharp waves,sharp slow waves,and multiple spasms with a series of attacks.High-throughput sequencing and Sanger sequencing identified a heterozygous mutation,c.5885 G>A(p.R1962H),in the DYNC1H1 gene(NM 001376)of the proband,which was not identified in her parents.Combined with the clinical manifestations and pedigree of this family,this mutation is likely pathogenic based on the American Academy of Medical Genetics and Genomics guidelines.The child was followed when she was 1 year and 2 mo old.The magnetic resonance imaging result was consistent with the findings of white matter myelinated dysplasia and congenital giant gyrus.The extensive neurogenic damage to the extremities was considered,as the results of electromyography showed that the motor conduction velocity and sensory conduction of the nerves of the extremities were not abnormal,and the degree of fit of the children with severe contraction was poor.At present,the child is 80 cm in length and 9 kg in weight,with slender limbs and low muscle strength,and still does not raise her head.She cannot sit or speak.Speech,motor,and mental development was significantly delayed.There is still no effective treatment for this disease.CONCLUSION We herein report a de novo variant of DYNC1H1 gene,c.5885 G>A(p.R1962H),leading to overlapping phenotypes(seizure,general growth retardation,and muscle weakness)of CMT20,MRD13,and SMA-LED,but there is no effective treatment for such condition.Our case enriches the DYNC1H1 gene mutation spectrum and provides an important basis for clinical diagnosis and treatment and genetic counseling.
基金Supported by Key Specific Program for Science and Technology of Guangdong Province (2008B020700003 A2007A020400006)~~
文摘[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.
文摘Age-related macular degeneration(AMD) is the leading cause of irreversible blindness in the developed world. The quality of life of both patients and families is impacted by this prevalent disease. Previously, macular degeneration had no known effective treatment. Today, vitamins for non-exudative AMD and intravitreal injection of medications for its exudative form are primary forms of current treatment. Modern advances in molecular science give rise to new possibilities of disease management. In the year 2003 the sequencing of the entire human genome was completed. Since that time, genes such as complement factor H, high-temperature requirement factor A1, and age-relateed maculopathy susceptibility 2 have been discovered and associated with a higher risk of AMD. A patient's genetic make-up may dictate the effectiveness of current or future therapeutic options. In addition, utilizing genetic data and incorporating it into new treatments(such as viral vectors) may lead to longer-lasting(or permanent) VEGF blockade and specific targeting of complement related genes. There have also been considerable advances in stem cell directed treatment of AMD. Retinal pigment epithelial(RPE) cells can be derived from human embryonic stem cells, induced pluripotent stem cells, or adult human RPE stem cells. Utilizing animal models of RPE and retinal degeneration, stem cell-derived RPE cells have been successfully implanted into the subretinal space. They have been injected as a cell mass or as a pre-prepared monolayer on a thin membrane. Visual recovery has been demonstrated in a retinal dystrophic rat model. Preliminary data on 2 human subjects also demonstrates possible early visual benefit from transplantation of stem cell-derived RPE. As more data is published, and as differentiation and implantation techniques are optimized, the stabilization and possible improvement of vision in individuals with non-exudative macular becomes a real possibility. We conclude that the technologic advances that continue to unfold in both genetic and stem cell research offer optimism in the future treatment of AMD.
文摘目的总结1个以下肢受累为主的脊髓性肌萎缩症(spinal muscular atrophy with lower extremity predominant,SMALED)家系的临床、电生理及影像特点,并对该家系进行致病突变分析。方法收集2020年8月首都医科大学宣武医院确诊为常染色体显性遗传的1例SMALED患者的家系资料。采集先证者及其女儿的临床资料,进行血常规、肝功能、肌酸激酶等检验,肌电图和神经传导检查,脊髓、头颅、双下肢MRI检查,表型匹配分析,对先证者进行SMN1基因拷贝数检测和全外显子组测序分析。按照美国医学遗传学与基因组学学会(American College of Medical Genetics and Ge-nomics,ACMG)和美国分子病理学会(Association for Molecular Pathology,AMP)基因变异解读标准和指南进行致病性分析,对先证者及其女儿进行Sanger验证。结果家系调查发现,先证者及其女儿表型与SMALED表型匹配。两人均存在DYNC1H1基因(c.1792C>T;p.R598C)杂合突变,根据ACMG和AMP指南考虑为强致病性突变。结论在2例患者中鉴定出DYNC1H1基因p.R598C的致病性突变,符合常染色体显性遗传方式,提示对中国人群SMALED的研究需进行DYNC1H1基因检测。
基金National "11th Five-year Plan" Scientific and Technical Supporting Programs (2006BAD06A11)
文摘Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.
文摘The influenza A viruses have three gene segments, M, NS, and PB1, which code for more than one protein. The overlapping genes from the same segment entail their interdependence, which could be reflected in the evolutionary constraints, host distinction, and co-mutations of influenza. Most previous studies of overlapping genes focused on their unique evolutionary constraints, and very little was achieved to assess the potential impact of the overlap on other biological aspects of influenza. In this study, our aim was to explore the mutual dependence in host differentiation and co-mutations in M, NS, and PB1 of avian, human, 2009 H1N1, and swine viruses, with Random Forests, information entropy, and mutual information. The host markers and highly co-mutated individual sites and site pairs (P values < 0.035) in the three gene segments were identified with their relative significance between the overlapping genes calculated. Further, Random Forests predicted that among the three stop codons in the current PB1-F2 gene of 2009 H1N1, the significance of a mutation at these sites for host differentiation was, in order from most to least, that at 12, 58, and 88, i.e., the closer to the start of the gene the more important the mutation was. Finally, our sequence analysis surprisingly revealed that the full-length PB1-F2, if the three stop codons were all mutated, would function more as a swine protein than a human protein, although the PB1 of 2009 H1N1 was derived from human H3N2.
基金supported by the major national S&T projects for infectious diseases(2018ZX10301401)the Key Research&Development Plan of Zhejiang Province(2019C04005)the National Key Research,and the Development Program of China(2018YFC2000500).
文摘The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.
基金Supported by National Natural Science Foundation of China (30721063)National Basic Research Program of China (973 Program) (2005CB522402, 2006CB910403)+1 种基金National Laboratory of Medical Molecular Biology grant (2060204)Beijing municipal government grant (YB20081002301)
文摘Objective To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. Methods HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls. Results Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells. Conclusion SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.
基金funded by the National Natural Science Foundation of China(31471176)the Fundamental Research Funds for the Central Universities,China(15054034)
文摘H5N1 influenza represents one of the great challenges to public health.Some H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to their natural hosts.Here,we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus.We demonstrated that,compared to the GS/65 virus,the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways(i.e.,RIG-I and MDA5) in ducks.We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes,while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus.We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis(WGCNA).Moreover,we first demonstrated that the duck BCL2 L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently.These analyses,in combination with our comprehensive transcriptomic data,provided global view of the molecular architecture for the interaction between host and H5N1 viruses.
文摘Objective: The nm23 gene is one of the tumor metastatic suppressor genes. The expression of nm23H1 has been reported to be inversely associated with metastatic potentiality in a number of human carcinomas, including breast, colorectal, gastric, hepatocellular and gallbladder carcinomas. In this study, the immunohistochemical staining of nm23H1 protein in human nasopharyngeal carcinoma (NPC) was examined, and the relationship between nm23H1 and both metastasis and prognosis of patients with NPC was also investigated. Methods: Routine LSAB immunohistochemistry with the nm23H1 monoclonal murine antibody was employed to study the expression of nm23H1 protein in 95 paraffinembedded specimens of NPC treated at our hospital. The clinical pathologic data and results of followup were also retrieved. Comparisons between patients with and without expression of nm23H1 protein with respect to metastasis, locoregional recurrence and survival were performed using Log rank test. Multivariate prognostic analyses were performed by using Cox's regression model. Results: Nm23H1 negative expressive tumors were associated with a higher incidence of lymphnode metastasis (86.7%) than those of nm23H1 positive (48.6%, P<0.01). Nm23H1 negative expressive tumors were associated with a high incidence of recurrence and distant metastasis after radiotherapy (P<0.05). A significant association was found between expression of nm23H1 and prognosis (P<0.01). The expression of nm23H1 indicated favorable prognosis. Conclusion: It was suggested that nm23 H1 negative expression was significantly associated with lymphnode metastasis, recurrence and distant metastasis. Nm23H1 may have value for predicting the prognosis of NPC.
基金supported by the Chinese National S&T Plan(2004BA519A55)Scientific Research Program of State Key Laboratory of Veterinary Biotechnology(NKLVBP200818)
文摘To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID<sub>50</sub> of the rAd-H5HA- EGFP was assessed to be 2.26×10<sup>10</sup>/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.
文摘In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.
