Magnetic resonance imaging focused on the ferritin heavy chain 1 reporter gene detects neuronal differentiation in stem cells

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders.Thus,there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells.Our previous study confirmed that magnetic resonance imaging,with a focus on the ferritin heavy chain 1 reporter gene,could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene.However,we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal.To solve this problem,we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells.In this study,we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene;we used this lentivirus to transduce bone marrow mesenchymal stem cells.Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells;this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal.In summary,we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter.This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells,which may be useful in stem cell-based therapies.

γANDδCHAIN GENE REARRANGEMENT OF T CELL RECEPTOR IN ACUTE LYMPHOBLASTIC LEUKEMIA

The immunophenotype, rearrangements of T cell receptor(TCR) γ andδchain genes as well as the immunoglobulinheavy chain (IgH)gene were studied in 37 cases ofmorphologically defined acute lymphoblastic leukemia (ALL).According to the expression of differentiation antigens, 8 caseswere classified as T-ALL, 26 B lineage ALL, 2 acute un-differentiated leukemia (AUL) and myeloid phenotype. An or-der of TCR gene rearrangements was observed in T-ALL,with the rearrangement of δgene preceding that of γgene.Both genes were also found frequently rearranged and / or de-leted in high proportions of the ALL of B cell lineage. Howev-er, the patterns of gene rearrangements were somewhat differ-ent between the T and B lineage ALLs. In contrast, the lgHgene rearrangements were observed only in the B lineage ALL.The immunogenotype analysis of ALL proved to be a usefulmarker of the clonality and provided us with important informa-tion on early human lymphoid differentiation. We concludethat the determination of T

Application of Landscape Gene Information Chain in Tourism Development of Traditional Human Settlements:A Case Study of Daqitou Village in Sanshui District,Foshan City

Based on the aspect of historical and cultural geography, the value of "landscape gene information chain" in the tourism development of ancient villages was verified by taking cultural landscape as the principal line, historical and cultural settlement as the carriers, and traditional buildings as the entry points. Taking Daqintou Village in Sanshui District, Foshan City for example, the theory of "landscape gene information chain" was applied to design a tourism planning scheme for Daqitou Village.

Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

AIM To study the significance of C-erbB-2 oncogene amplification in gastric cancer.METHODS C-erbB-2 oncogene amplification was examined by using differential polymerase chain reaction (dPCR) in surgical and endoscopic specimens of 83 cases of gastric cancer and 101 metastatic lymph nodes.RESULTS C-erbB-2 amplification was found in 28.9% (24/ 83) surgical specimens and 20.5% (17/ 83) endoscopic ones of gastric cancer patients. The amplification was significant in both types of specimens of advanced cancer cases (P＜0.05) and surgical specimens with lymph node metastasis (P＜0.01). The incidence of C-erbB-2 amplification in lymph nodes with metastasis was higher than in primary sites (surgical specimens, P＜0.05). The patients with amplification tumors had poorer 5-year survival rates than those with unamplification ones in the early cancers and well to moderately differentiated adenocarcinomas (P＜0.05). The same surgical samples were tested again by Southern blot hybridization to ascertain C-erbB-2 amplification, and the positive rate of C-erbB-2 amplification (15.7%) was lower than that of dPCR (28.9%, P＜0.05).CONCLUSION Examining C-erbB-2 amplification by dPCR is a quick, simple, reliable and independent method, and is helpful in predicting prognosis and metastatic potential of gastric cancer.

CHARACTERIZATION OF THE β－MYOSIN HEAVY CHAIN GENE MISSENSE MUTATIONIN A CHINESE PATINENT WITH HYPERTROPHIC CARDIOMYOPHTHY

We have analyzed the exons 13, 16, 21 and 23 of cardiac myosin heavy chain gene in 32 Chinese patients with hypertrophic cardiomyopathy by using PCR-single strand conformation polymorphism (PCR-SSCP) procedure. The result showed an altered SSCP of the exon 13 in one patient. Sequencing analysis revealed that the patient had a G to T transversion in the codon 383, resulting in the substitution of Lys by Asn. Beacause the missense mutation was found at the residue highly conserved through species evolution, this mutation is likely, to be the cause of hypertrophic cardiomyopathy in this patient. This is the first report of a mutant cardiac β-MHC gene in the Chinese population. Also, it is a novel missense mutation of the cardiac β-MHC gene.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Two－step multiplex polymerase chain reaction for gene diagnosis of progressive pseudohypertrophic muscular dystrophy

in the present study,9 exon-containing DNA segments of dystrophin gene with 9 sets of oligonucleotide primers by two-step multiplex polymerase chain reaction (mPCR) were amplified. Subsequently,gene analysis was performed in 36 cases of Duchenne mascular dystroply (DMD) and 4 cases of Becker muscular dystrophy(BMD). The findings showed that 17 cases of deletion were detected by using the first 5 sets of primers with a relatively high incidence of deletion detection and 2 more cases of deletion were detected by using the remaining 4 sets of primers. The total deletion rate detected by mPCR with 9 cases of primers was 47. 5% of the patients examined,suggesting that about 79. 1% of the patients with gene deletion could be detected. Thus,as a preliminary screening, the two-step mPCR can be used in the gene diagnosis of DMD/BMD. The method is not only simple, convenient and rapid,but also free from radiosotope trouble.

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.

Insertional mutagenesis and cloning of HIV env 125 peptide gene by polymerase chain reaction

One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...

Construction and Control of Genetic Regulatory Networks:A Multivariate Markov Chain Approach

In the post-genomic era, the construction and control of genetic regulatory networks using gene expression data is a hot research topic. Boolean networks (BNs) and its extension Probabilistic Boolean Networks (PBNs) have been served as an effective tool for this purpose. However, PBNs are difficult to be used in practice when the number of genes is large because of the huge computational cost. In this paper, we propose a simplified multivariate Markov model for approximating a PBN The new model can preserve the strength of PBNs, the ability to capture the inter-dependence of the genes in the network, qnd at the same time reduce the complexity of the network and therefore the computational cost. We then present an optimal control model with hard constraints for the purpose of control/intervention of a genetic regulatory network. Numerical experimental examples based on the yeast data are given to demonstrate the effectiveness of our proposed model and control policy.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

猪萨佩罗病毒聚合酶链式反应检测方法的建立及应用

猪萨佩罗病毒(Porcine sapelovirus,PSV)是引发猪腹泻的重要病毒之一。为开发一种能快速、准确检测PSV的聚合酶链式反应检测方法,针对PSV的保守区3D基因设计了一对特异性引物,优化了聚合酶链式反应退火温度,分析聚合酶链式反应检测方法特异性和灵敏度,并应用于猪场样品检测。结果表明:建立的聚合酶链式反应检测方法可特异性扩增出PSV目的片段,对其他非靶标猪腹泻病毒则无条带产生,检测低限为5拷贝/μL;在浙江地区采集的88个健康猪粪便样本中检测PSV阳性率为7.95%。该PSV聚合酶链式反应检测方法特异性强、灵敏度高,可应用于PSV前期诊断和防控。

土壤固碳微生物的绝对定量检测实验设计

为了定量检测土壤中的固碳微生物,设计以功能基因cbbL为靶标的固碳微生物微滴数字聚合酶链式反应(ddPCR)检测方法。选择合适的引物探针,从退火温度、探针浓度以及引物浓度进行反应条件的优化,分析ddPCR检测方法的线性范围、敏感性、重复性和特异性。结果显示,当退火温度为55.8℃、探针与引物浓度分别为350、750 nmol/L时,建立的cbbL-ddPCR扩增反应效率最高,阴阳性微滴分布界限最明显,平均拷贝数较高;检测的线性范围为2.3×10^(0)~2.3×10^(5)copies/μL-DNA,曲线方程y=0.1077x-95.562,相关系数R^(2)为0.9997,检出限为0.5 copy/μL-DNA,21个重复的变异系数仅为3.92%,与其他4种非固碳微生物DNA未发生交叉反应。所建立的cbbL-ddPCR方法可用于土壤微生物固碳潜能测定。

Epidemiology and molecular genetics of congenital cataracts

Congenital cataract is a crystallin severe blinding disease and genetic factors in disease development are important. Crystallin growth is under a combination of genes and their products in time and space to complete the coordination role of the guidance. Congenital cataract-related genes, included crystallin protein gene (CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS), gap junction channel protein gene (GJA1, GJA3, GJA8), membrane protein gene (GJA3, GJA8, MIP, LIM2), cytoskeletal protein gene (BF-SP2), transcription factor genes (HSF4, MAF, PITX3, PAX6), ferritin light chain gene (FTL), fibroblast growth factor (FGF) and so on. Currently, there are about 39 genetic loci isolated to which primary cataracts have been mapped, although the number is constantly increasing and depends to some extent on definition. We summarized the recent advances on epidemiology and genetic locations of congenital cataract in this review.

急性缺血性脑卒中患者血清长链非编码RNA母系表达基因3和Zeste同源物增强子-2表达与神经功能损伤的相关性分析

目的探讨长链非编码RNA(lncRNA)母系表达基因3(MEG3)与Zeste同源物增强子-2(EZH2)在急性缺血性脑卒中(AIS)患者血清中的表达水平及其与神经功能损伤之间的关系。方法选取延安市人民医院2021年1月至2022年11月收治的92例AIS患者为脑卒中组,92例健康体检者为对照组,根据NIHSS评分将脑卒中组患者分为致残性损伤组19例,非致残性损伤组73例。采用实时荧光定量聚合酶链式反应、酶联免疫吸附法分别检测血清lncRNAMEG3、EZH2水平。采用Spearman相关分析法进行AIS患者血清lncRNAMEG3、EZH2表达水平与美国国立卫生研究院卒中量表(NIHSS)评分之间的相关性分析;采用多因素Logistic回归分析AIS患者合并神经功能致残性损伤的影响因素,并绘制受试者工作特征(ROC)曲线分析血清lncRNA MEG3、EZH2水平对AIS患者合并神经功能致残性损伤的诊断价值。结果脑卒中组患者血清lncRNA MEG3、EZH2表达水平均高于对照组(t=11.817、11.542,P<0.05)。Spearman相关分析显示,AIS患者血清lncRNA MEG3、EZH2水平与NIHSS评分均呈正相关(r=0.540、0.603,P<0.01)。致残性损伤组体质量指数、吸烟史占比、甘油三酯、总胆固醇、同型半胱氨酸、发病-到院时间(ODT)、lncRNAMEG3、EZH2水平均高于非致残性损伤组(t/χ2=2.103、5.050、9.121、5.585、2.276、5.448、4.638、4.682,P<0.05)。多因素Logistic回归分析显示,甘油三酯、总胆固醇、lncRNA MEG3、EZH2以及ODT均是AIS患者合并神经功能致残性损伤的影响因素(OR=4.853、4.277、2.674、3.052、3.901,P<0.05)。lncRNA MEG3、EZH2联合诊断AIS患者合并神经功能致残性损伤的曲线下面积(AUC)大于lncRNAMEG3以及EZH2单独诊断的AUC(Z=2.626、2.954,P<0.01),敏感度、特异度分别为94.74%、82.15%。结论AIS患者血清lncRNA MEG3、EZH2水平均上升,与AIS患者神经功能损伤程度均正相关,可用作AIS患者合并神经功能致残性损伤的诊断指标,且联合诊断效果更好。

仰口线虫PCR检测方法的建立及应用

为特异性检测羊感染仰口线虫的情况,基于仰口线虫ITS基因序列设计特异性引物,进行退火温度及反应条件的优化,建立其属特异性PCR检测方法。应用该方法对仰口线虫、食道口线虫、毛尾线虫、捻转血矛线虫、细颈线虫、奥斯特线虫、夏伯特线虫、筒线虫、毛圆线虫的DNA样本进行扩增,仅能特异性扩增出仰口线虫的目的条带;该方法成功从仰口线虫中扩增出约500 bp的特异性片段,最低检测浓度为3.6×10^(3)copies/μL;该PCR检测方法对临床奶山羊粪便样品的检测结果显示,羊粪便样品中仰口线虫卵的阳性率为10%,且与显微镜镜检所得的结果一致。结果表明,建立的仰口线虫PCR检测方法敏感性较高且特异性良好,可以应用于临床羊粪便样品中仰口线虫的虫卵检测,为羊仰口线虫病的诊断与防治提供了一种技术支持。

基于连接酶链式反应的EML4-ALK融合基因分型检测

针对棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶(EML4-ALK)融合基因变异体V1、V2、V3a、V3b设计引物、探针,优化连接探针长度及其浓度,建立基于连接酶链式反应的qPCR法检测方案,并对该方案的特异性、检出限、抗干扰性进行验证。结果表明:基于连接酶链式反应的qPCR法能对EML4-ALK融合基因进行有效分型;连接探针长度在30 nt(nucleotide)时检测灵敏度、特异性最高;连接探针终浓度在0.1~1.0 nmol/L较为合适;设计的引物、探针特异性好,只扩增靶RNA,对其他RNA无扩增;对EML4-ALK融合基因变异体V1、V2的检出限为10 copies/μL,变异体V3a、V3b的检出限为100 copies/μL;在EML4-ALK融合基因RNA标准品检测过程中加入100 ng的肺癌样本RNA作为干扰,检测结果未受到明显干扰。

景观基因信息链下江头村公共设施优化设计

文章基于景观基因信息链理论,对江头村景观基因进行挖掘梳理,构建江头村风貌景观基因图谱,探索公共设施的优化设计策略。将物质风貌景观基因与非物质风貌景观基因进行分析与提取,以造型、色彩、纹样、材料、工艺、信仰及习俗七大因素为依据,构建江头村风貌景观基因图谱和风貌景观基因评价模型树,再使用层次分析法(AHP)对所提取的7项基因进行判断矩阵赋权,根据权重计算结果指导公共设施的优化设计。对公共设施的优化设计不仅满足了基本功能的实用需求,还提升了村落的文化价值,增强了人们对江头村景观风貌的认同感与归属感。