Allele-specific polymerase chain reaction (AS-PCR) is a technique based on allele-specific primers, which can be used to analyze single nucleotide polymorphism (SNP) effectively including the transition, transversion and insertion/deletion polymorphism and has been exploited in the study of diseases research, molecular diagnosis, and forensic biological evidence. The article systematically reviews the principle, the detection methods, improvement of AS-PCR, and its research updates in the fields of autosome, Y chromosome and mitochondrial SNP, as well as its application in forensic science.
Alleles ; DNA Primers ; Forensic Sciences ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate the effects of different concentrations of lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), dexamethasone (Dex), and insulin on the mRNA and protein expressions of GPR54 in the MCF7 cell line in vitro.

METHODSMCF7 breasr cancer cells were cultured and treated with different concentrations of LPS (10 and 20 µg/ml), TNFα (20 and 100 ng/ml), IL-6 (10 and 20 ng/ml), Dex (10(-6) and 10(-7) mol/L), and insulin (0.01 and 0.1 IU/L). Those treated with culture fluid only served as controls. The mRNA and protein expressions of GPR54 were measured by real-time PCR and Western blot, respectively, after 6, 24, 48, and 72 hours of treatment.

RESULTSCompared with the blank con- trol, LPS (10 and 20 µg/ml), TNFα (20 and 100 ng/ml), IL-6 (10 and 20 ng/ml), Dex (10(-6) and 10(-7) mol/L), and insulin (0.01 and 0.1 IU/L) significantly increased the expressions of GPR54 mRNA (P < 0.05) and protein (P < 0.05).

CONCLUSIONLPS, TNFα, IL-6, Dex, and insulin evidently increase the expression of GPR54 in the MCF7 cell line, indicating their influence on the function of gonads by regulating the GPR54 level.

Blotting, Western ; Dexamethasone ; administration & dosage ; pharmacology ; Glucocorticoids ; administration & dosage ; pharmacology ; Gonads ; drug effects ; metabolism ; Humans ; Hypoglycemic Agents ; administration & dosage ; pharmacology ; Insulin ; administration & dosage ; pharmacology ; Interleukin-6 ; administration & dosage ; pharmacology ; Lipopolysaccharides ; administration & dosage ; pharmacology ; MCF-7 Cells ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, G-Protein-Coupled ; drug effects ; genetics ; metabolism ; Receptors, Kisspeptin-1 ; Time Factors ; Tumor Necrosis Factor-alpha ; administration & dosage ; pharmacology

This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 microg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75% -100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 microg/mL concentration. Both 25 microg/mL and 50 microg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 microg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2 * WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.
Animals ; Bone Marrow Cells ; Cell Differentiation ; Dextrans ; chemistry ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Polylysine ; chemistry ; Rats ; Staining and Labeling

Ulcerative colitis (UC) is a chronic inflammatory disease and its involvement area in colon is influenced by a complex network of gene interactions.We analyzed the weighted gene co-expression networks in microarray dataset from colonic mucosa of patients with UC and identified one gene co-expression module that was highly associated with the progression of involved area in UC colon (Pearson coefficient=0.81,P＜0.0001).In total,523 hub genes in this module were found to be involved in immune system process after enrichment analysis in Gene Ontology.By the STRING and Cytoscape analysis,we observed that interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) were centered in the network of hub genes.We then detected the expression of IL-8 and MMP-9 in mucosa from left-sided colon of patients using quantitative PCR and immunofluorescence assay respectively.Both quantitative PCR and immunofluorescence assay revealed the expression levels of IL-8 and MMP-9 were significantly different among the healthy controls,left-sided colitis group and pancolitis group (P＜0.05).IL-8 and MMP-9 were detected with an enhanced expression in pancolitis as compared with left-sided colitis and healthy controls,respectively (P＜0.05).This study demonstrates that immune system process is indispensable in the progression of disease in colon,and identifies that IL-8 and MMP-9 play potential critical roles for the progression.

Objective To study the relationship between level of plasma glucose and cognitive function in patients with Parkinson's disease.Methods Two hundred PD patients were assessed cognitive function using Mini-Mental State Examination (MMSE),Montreal Cognitive Assessment (MoCA),Wechsler Intelligence Scale and Wechsler Memory Scale.The patients were divided into cognitive normal group (n=91) and cognitive impairment group (n=109).One hundred twenty-six normal subjects were enrolled as control group (n=126).The levels of fasting plasma glucose (FPG),postprandial plasma glucose (2hPPG),glycosylated hemoglobin (HbAlc) and the prevalence of diabetes mellitus were compared among the groups.The effect of blood glucose level on the cognitive function of PD patients was analyzed by Binary Logistic Regression.Results The levels of FPG,HbAlc and the prevalence of diabetes mellitus [5.19 (0.72),5.7％ (0.5％),14％] were significantly higher than those in the normal control group [4.85(0.79),5.6％ (0.5％),6％] (P＜0.05).The levels of FPG in PD patients with cognitive impairment [5.21 (1.32)] was significantly higher than that in PD patients with cognitive normal group [4.81 (0.95)] (P＜0.05).Although 2hPPG and HbAlc increased slightly in PD patients with cognitive impairment,the difference did not reach an significant level (P＞0.05).Binary Logistic Regression analysis showed that FPG(OR:1.764;95％ CI:0.06-3.244;P=0.068) was not associated with the impaired cognitive function in PD patients.Conclusion The present study has not revealed an association between the incidence of cognitive impairment in patients with PD and plasma glucose level although high plasma glucose may be a high risk factor for PD patients.

OBJECTIVETo clone a novel gene expressed specifically in human embryonic stem cell and to analyze its characteristics.

METHODSBased on an expression sequence tags(EST) CF948547 which expressed specifically in human embryonic stem cell, the full-length cDNA sequence of a novel gene was cloned by using bioinformatic and molecular biological technique. Its expression profile was analyzed by reverse transcription-polymerase chain reaction(RT-PCR), and subcellular location was determined by enhanced green fluorescent protein (EGFP) eukaryotic expression system.

RESULTSA novel gene HPESCRG1(homo sapiens pluripotent embryonic stem cell-related gene) was cloned successfully. Its GenBank accession number was AY283672. Its cDNA length was 1395 bp. It comprised 9 exons and 8 introns, and its opening reading frame was 250-1146 bp. Its chromosomal mapping was located in 3q13.13, and the putative protein contained 297 amino acids. The theoretical molecular weight of the putative protein was 33 784 and the isoelectric point was 9.35. The protein primary structure of this gene contained a SAP motif and it was subcellularly located in nuclei. Expression analysis showed that this gene was expressed specifically in human ES cells, but not expressed in the adult human tissues, the multiple tissues of embryo aborted in over 5 months' pregnancy, the differentiated cells of HESC-1, and the human mesenchymal stem cells (hMSCs) and human embryonic fibrocytes (hEFCs).

CONCLUSIONHPESCRG1 was found to be a novel gene expressed specifically in human ES cell, which might be related to self-renewal of human ES cell and maintaining its undifferentiated state.

Amino Acid Sequence ; Base Sequence ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; analysis ; Embryo, Mammalian ; Exons ; Expressed Sequence Tags ; Gene Expression ; Humans ; Introns ; Molecular Sequence Data ; Molecular Weight ; Nuclear Proteins ; Open Reading Frames ; genetics ; Proteins ; genetics ; metabolism ; Stem Cells ; cytology ; metabolism

Objective:To explore the experience of self-management dilemma ofadults with emerging ankylosing spondylitis, and to provide reference for the construction of self-management intervention strategies for emerging adults with ankylosing spondylitis.Methods:Descriptive phenomenology was used to conduct in-depth interviews with 14 adults with emerging ankylosing spondylitis in the Rheumatology and Immunology Department of Drum Tower Hospital Affiliated to Medical College of Nanjing University from August 2022 to March 2023. The interview data were analyzed by Colaizzi′s seven-step analysis method.Results:A total of 14 patients completed the interview,10 males, 4 females, aged 21-30 years. In adults with emerging ankylosing spondylitis, there were dilemmas of role maladjustment and disease management disorder, including role maladjustment of disease management and social role maladjustment. Barriers to disease management included weak self-management awareness, insufficient support for self-management information, inadequate self-management skills, and poor compliance with self-management behaviors.Conclusions:The role adaptation and self-management ability of adults with emerging ankylosing spondylitis are seriously inadequate. It is urgent to construct health management strategies for adults with emerging ankylosing spondylitis to help them improve the level of role adaptation and disease management.

OBJECTIVE: To investigate the application of dinucleotide STR locus in paternity testing. METHODS: Dinucleotide STR locus D6S261 was selected and the paternity testing blood samples were amplified using 200 random blood samples, 16 family samples and 193 paternity test samples. Data of the PCR products were collected by 3130XL Genetic Analyzer and the genetic parameters of population were calculated by PowerStats v12. RESULTS: Fifteen alleles and 50 genotypes were found and H, DP, PE and PIC were 0.850, 0.953, 0.695, and 0.820, respectively. The typing results of both family samples and paternity test samples were accord with the law of inheritance, which no mutation was discovered. CONCLUSION The genetic polymorphisms of D6S261 show good characteristics with low mutation rate and high stability. It can be an effective method to solve the indetermination caused by mutation in paternity testing if the stutter bands can be decreased.
Asian People/genetics* ; Base Sequence ; Forensic Genetics/methods* ; Gene Frequency ; Genotype ; Humans ; Microsatellite Repeats/genetics* ; Nucleotides/genetics* ; Paternity ; Polymerase Chain Reaction/methods* ; Polymorphism, Genetic

In order to evaluate the immunogenicity of HPV 58 L1 DNA vaccines, five DNA vaccines had been constructed with pcDNA3.1 vector containing different L1 genes of HPV 58, which were designated as L1h, L1hDeltac, L1S, L1SM and L1wt. The protein expression of DNA vaccines in vitro was tested by Western blot. The ability of forming pseudovirus was evaluated by transfecting DNA vaccine together with pcDNA3.1-h58L2 and pcDNA3.1-GFP into 293FT cells. The neutralizing antibodies and cellular immune response produced in BALB/c mice immunized with the DNA vaccines were detected by using pseudovirus-based neutralization assay and ELISPOT respectively. The results showed that the five DNA vaccines had been successfully constructed; the level of protein expression of L1hDeltac was the highest and those for L1S and L1SM were of medium, while no expressed target protein of L1wt was detected. Only L1S could form the pseudovirus while the other four vaccines could not. L1S and L1h could induce neutralizing antibody. However, the average titer of neutralizing antibody for L1S (1:6,400) was much higher than that for L1h (1:48) and the other three vaccines could not induce neutralizing antibody. No cellular immune response for all five DNA vaccines was detectable by ELISPOT. The results indicated that DNA vaccine against HPV 58 can form pseudovirus in vitro, also can induce high level of neutralizing antibodies. This provides reference for screening HPV vaccine in future.
Animals ; Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Models, Genetic ; Neutralization Tests ; Papillomaviridae ; immunology ; Papillomavirus Vaccines ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Vaccines, DNA ; genetics ; immunology

This study was aimed to explore the clinical features and prognosis outcome of childhood T-cell acute lymphoblastic leukemia (T-ALL). The clinical data of 38 cases of newly diagnosed T-ALL from Jan 2005 to Aug 2010 were analyzed retrospectively, and 78 cases of B-ALL with intermediate and high risk were collected as control group, then the sensitive rate of patients to prednisone pretreatment, complete remission (CR) rate at day 33 after induction chemotherapy, relapse rate and 3-year event-free survival (EFS) were compared between T-ALL and B-ALL children. The results showed that no significant statistic difference were found in distribution of age, infiltration of liver, spleen and lymph nodes as well as central nervous system disease, chromosome abnormality, expression level of fusion gene and so on between T-ALL and B-ALL groups (p > 0.05), but there were significant differences in sex and number of cases with WBC count ≥ 50 × 10(9)/L between them (p < 0.05). The sensitive rate of T-ALL and B-ALL patients to prednisone pretreatment was 51.9% and 89.3% respectively (p < 0.05). The ratio failed to achieve CR at day 33 after induction chemotherapy was 15.4% and 8.1% in the two groups (p > 0.05). The relapse rate of T-ALL and B-ALL cases was 30.8% (8/26) and 14.9% (11/74) respectively (p > 0.05). The time from CR to relapse was (9.78 ± 3.48) month and (21.28 ± 14.32) month (p < 0.05). The 3 year EFS of T-ALL cases with intermediate and high risk was (37.5 ± 17.1)% and (22.2 ± 9.8)%, while 3 year EFS of B-ALL cases was (66.7 ± 7)% and (51.7 ± 9.3)% respectively (p < 0.05) according to Kaplan-Meier survival curve. It is concluded that as compared with B-ALL cases, the male ratio and initial WBC count are higher, moreover the early response to prednisone pretreatment and 3 year EFS are poor in T-ALL cases, the prognosis outcome is poor also.
Adolescent ; Child ; Child, Preschool ; Disease-Free Survival ; Female ; Humans ; Immunophenotyping ; Infant ; Male ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; mortality ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; mortality ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; immunology ; mortality ; Prognosis ; Retrospective Studies ; Survival Rate