Colon cancer is the most common malignant tumor in the worldwide scale and it is one of the leading causes of cancer-related deaths.Finding accurate and informative cancer markers will provide significant insights in diagnosis and treatment strategy of colon cancer.Proteomic technology is an important tool in cancer research.In recent years,many studies have found a great of potential biomarkers of colon cancer.

Objective:Based on the high-throughput detection technique of multiplex PCR combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, constructing the characteristic SNP profiles of different strains, and establishing a rapid, accurate and highly sensitive method for the diagnosis of bloodstream infection pathogens.Methods:Seven kinds of pathogens such as common Escherichia coli were selected as target. The multiple PCR reaction conditions was optimized, and the characteristic peaks of each target bacteria were detected by MALDI-TOF MS to establish the joint detect system. Common primer pairs and central homo-sequence primer pairs were designed to analyse the formation of primer dimer. Using simulated bacterial infection blood samples with detection system to determine specificity and sensitivity. One hundred and fifty blood samples from suspected bacteremia patients were collected from June to September 2020 in a hospital in Beijing, and the identification results were compared to traditional identification method of clinical application that are using χ 2 test. Results:The cycle threshold (Ct) value of the central homo-sequence primers that were designed were more than 38, with a delay of 6-10 cycles. The joint mass spectrometry detection system could detect seven kinds of bacteria divided into two groups at the same time. The target bacteria can be detected specific product of the peak, and the clinical strains other than the target strains only had primer peaks. All maps had non-specific miscellaneous peaks. The sensitivity of Escherichia coli could reach 50 CFU/ml, and the detection limit of other bacteria was 100 CFU/ml. The detection results of 150 patients showed that 46 cases were positive by traditional method. The positive rate was 30.67% (46/150), including two cases of mixed infection. Forty-eight cases were positive by mass spectrometry, and the positive rate was 32.0% (48/150), including three cases of mixed infections. The negative coincidence rate was 100% (101/101). The comparison of the two methods showed that the P=0.625>0.01, the Kappa=0.938, the sensitivity and specificity was 97.82%(45/46) and 97.11%(101/104), respectively. There was no significant difference between the two methods, and the results of nucleic acid mass spectrometry could also be used in clinic. Conclusions:The established detection system can not only quickly and accurately detect seven common pathogens causing bloodstream infection, and effectively shorten the time needed for traditional culture and identification, but also can detect multiple bacterial mixed infections at the same time to make up for the possibility of missed detection. Besides, the method can also be used to identify other bacteria.

Objective To clone and express the outer surface protein C ( OspC) from a Chinese Borrelia afzelli FP1 strain and to evaluate the immune protectivity of the recombinant OspC protein ( rOspC) . Methods The gene encoding OspC protein of Borrelia afzelli FP1 strain was amplified by polymerase chain reaction (PCR) and then inserted into pET-30a plasmid to construct the recombinant expression plasmid pET-30a-OspC. The transformed E. coli BL21 strains carrying pET-30a-OspC plasmid were induced by IPTG to express OspC protein. The expressed proteins were purified by Ni-IDA resin chromatography and analyzed by SDS-PAGE and Western blot assay. Indirect immunofluorescence assay ( IFA) was performed to detect anti-rOspC protein antibodies in serum samples from rabbits immunized with rOspC protein. In vitro neutral-ization test was performed for evaluation the immune protectivity of rOspC protein. Results The recombi-nant expression plasmid pET-30a-OspC was successfully constructed and highly expressed in E. coli BL21. A strong antigen-antibody reaction between the rOspC protein and polyclonal antibody against Borrelia afzelli FP1 strain was detected by Western blot assay. The titers of IgG in serum samples from rabbits immunized with rOspC protein were significantly elevated. The in vitro neutralization test indicated that 106/ml of Borre-lia afzelli FP1 strains were neutralized by every anti-OspC protein serum sample from the experiment group. Conclusion The rOspC protein showed a strong immune protectivity against Borrelia afzelli, which could be used in the development of polyvalent subunit vaccine against lyme disease.

Objective To observe the effectiveness and mechanism of Xiaoyao San (Xiaoyao Powder for Soothing Liver and Relieving Depression) in improving depression-like behavior of rats. Methods Male Wistar rats were randomized into normal group, model group, Xiaoyao San (1.9 g·kg-1·d-1) group, and fluoxetine (2 mg·kg-1·d-1) group. The rats were exposed to chronic unpredictable mild stress ( CUMS) to induce rat depression-like behavior. Field test was performed for the observation of effect of Xiaoyao San on rat depression-like behavior, Luminex liquid chip system was applied to detect the serum cytokines, and the amount and size of rat hepatic sinusoidal endothelial window were examined under electron microscope, and hepatic indoleamine 2, 3-dioxygenase ( IDO) and tryptophan 2, 3 -dioxygenaes ( TDO) expression levels were detected by immunohistochemical and Western blot methods. Results Xiaoyao San showed obvious effect on increasing sugar water consumption, the number of crossing the blocks and erection frequency in rats, decreasing serum levels of tumor necrosis factor alpha ( TNF-α) and interleukin 6 ( IL-6) , increasing the amount of hepatic sinusoidal endothelial window, promoting hepatic sinusoidal endothelial vascularization, and reducing TDO and IDO expression ( P<0.05 or P<0.01). Conclusion Xiaoyao San exerts obvious effect on improving rat depression-like behaviors, and the mechanism is probably related with the decrease of inflammatory factors, inhibition of IDO pathway, and improvement of hepatic sinusoidal endothelial function.

OBJECTIVE: To explore the genetic etiology of two patients with Perrault syndrome (PRLTS) in a family. METHODS: Whole exome sequencing (WES) was carried out to screen potential variants within genomic DNA extracted from the proband. Suspected variants were validated by clinical data and results of Sanger sequencing. RESULTS: WES has identified two heterozygous variants of TWNK gene, namely c.1172G>A (p.Arg391His) and c.1844G>C (p.Gly615Ala). Sanger sequencing confirmed that the c.1172G>A (p.Arg391His), a known pathogenic variant, was derived from her father, while the c.1844G>C (p.Gly615Ala), a novel variant, was derived from her mother. Her brother, who was similarly affected, has carried the same compound heterozygous variants. CONCLUSION The compound heterozygous variants c.1172G>A (p.Arg391His) and c.1844G>C (p.Gly615Ala) of the TWNK gene probably underlie PRLTS in the sib pair. The above results have facilitated genetic counseling and prenatal diagnosis for the affected family.

Objective:To explore the clinical phenotype and genotype of a family with hereditary hypofibrinogenemia.Methods:Activated partial thrombin time (APTT), prothrombin time (PT),thrombin time (TT) and thrombelastogram (TEG) were tested in all family members. Fibrinogen activity and antigen were detected by Clauss method and immunoturbidimetric method respectively. All exons and flanking sequences of fibrinogen FGA,FGB,FGG genes were analyzed by PCR, and the products were subjected to Sanger sequencing.Results:The proband represented prolonged PT and TT, low Fg activity and antigen, elevated K value and decreased Angle value in TEG. Other family members reported similar changes including proband′s father,daughter and son, and his elder brother and his niece. Exon 5 c.510_512 of FGG gene in the proband revealed a minor deletion mutation.Conclusion:The novel heterozygous missense mutation of exon 5 c.510_512del (Gln170_Ile171 del ins His) of FGG gene is the molecular mechanism that leads to hereditary hypofibrinogenemia in this family.

OBJECTIVETo explore the genetic etiology of fetal abnormalities detected by prenatal ultrasound through single nucleotide polymorphism (SNP array) analysis.

METHODSTwo hundred and eight fetuses were tested with SNP array and conventional karyotyping. Complex copy number variations (CNVs) were verified with fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and quantitative fluorescence polymerase chain reaction (QF-PCR).

RESULTSFor the 208 cases, the diagnostic yields of conventional karyotping and SNP assay were 8.2%(17/208) and 13.9%(29/208), respectively. For fetuses with malformations of the cardiovascular system, central nervous system or multiple systems, pathogenic CNVs was detected in 4.6% (8/174), 2.3%(4/174), and 1.1% (2/174) of all fetuses, respectively. No pathogenic CNVs was detected among those with abnormalities of the renal system, digestive system, skeletal system, facial dysmorphism or respiratory system.

CONCLUSIONCNVs are significantly related with birth defects. Compared with conventional karyotyping, SNP array is a better platform for CNVs detection and can provide more clues for genetic counseling, recurrence risk assessment and prenatal diagnosis.

Adult ; Chromosome Aberrations ; DNA Copy Number Variations ; Female ; Fetal Diseases ; diagnosis ; diagnostic imaging ; genetics ; Genome-Wide Association Study ; Humans ; Infant, Newborn ; Karyotyping ; Male ; Polymorphism, Single Nucleotide ; Pregnancy ; Pregnancy Complications ; diagnosis ; diagnostic imaging ; genetics ; Prenatal Diagnosis ; Ultrasonography ; Young Adult

OBJECTIVETo explore the role of mitochondrial DNA 5178 C/A (Mt5178) polymorphism of NADH-dehydrogenase subunit 2 (ND2) gene in type-2 diabetes mellitus (T2DM) among ethnic Han Chinese through a case-control study.

METHODSThe Mt5178C/A polymorphism was determined by sequencing 1103 T2DM patients and 791 healthy controls. Logistic regression analysis was conducted to estimate odds ratios (OR) and 95% confidence intervals (CI). To confirm the results, a meta-analysis was conducted based on published literature on the association of Mt5178 variant with T2DM.

RESULTSNo significant association was found between the Mt5178C/A variant and T2DM either by our study or the meta-analysis which included eight published studies. Nevertheless, it was found that the T2DM patients with 5178C genotype were at a higher risk for nephropathy complication (OR=1.49, 95%CI: 1.005-2.197, P<0.05) and at significantly lower risk for hypertension complication (OR=0.744, 95%CI: 0.556-0.996, P<0.05) compared with those carrying a 5178A genotype.

CONCLUSIONNo association was found between the Mt5178C/A polymorphism of mitochondrial ND2 gene with the increased risk of T2DM. However, the polymorphism may affect the development of nephropathy and hypertension complications among T2DM patients.

Adult ; Aged ; Blood Glucose ; metabolism ; Cholesterol ; blood ; Cholesterol, HDL ; blood ; DNA, Mitochondrial ; chemistry ; genetics ; Diabetes Complications ; blood ; genetics ; Diabetes Mellitus, Type 2 ; blood ; complications ; genetics ; Diabetic Nephropathies ; blood ; genetics ; Fasting ; blood ; Female ; Humans ; Hypertension ; blood ; complications ; genetics ; Male ; Meta-Analysis as Topic ; Middle Aged ; Odds Ratio ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA ; Triglycerides ; blood

OBJECTIVETo report on a Chinese family from Wenzhou with genetically confirmed Kennedy disease and describe its clinical and genetic features.

METHODSThe clinical phenotype and the level of relevant biochemical markers were assessed. To determine the number of CAG repeats in the exon 1 of androgen receptor (AR) gene, genomic DNA was extracted from peripheral blood samples of the family members, amplified by PCR and identified by DNA sequencing.

RESULTSThe proband showed predominantly proximal limb weakness, fasciculation, muscle atrophy, gynecomastia, sexual dysfunction and increased serum creatine kinase. Myopathy and neuropathy were identified by electromyography. Two other affected males and 2 affected female carriers were identified to carry an expanded CAG repeat in the AR gene. The numbers of CAG repeats were found to be 43 in the proband, 43 and 42 in the other two affected males, one of which had similar clinical symptoms to the proband.

CONCLUSIONThe family was diagnosed with Kennedy disease by analysis of the AR gene.

Adolescent ; Adult ; Base Sequence ; Bulbo-Spinal Atrophy, X-Linked ; blood ; diagnosis ; genetics ; Creatine Kinase ; blood ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Pedigree ; Receptors, Androgen ; genetics ; Trinucleotide Repeat Expansion ; Young Adult

OBJECTIVETo develop a rapid, simple, cost-effective, accurate and sensitive method for quantitative detection of mitochondrial DNA (mtDNA) 3243A→G mutation in order to provide reference for selecting the best detection method under different conditions.

METHODSGenomic DNA was extracted from peripheral leucocytes of 17 individuals from a Wenzhou family featuring maternally inherited diabetes and deafness (MIDD). Heteroplasmic level of mtDNA 3243A→G mutation was determined respectively with polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP), real time-amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) and pyrosquencing. Eleven plasmids with various heteroplasmic levels of the 3243A→G mutation (ranging from 0 to 100%)were constructed as the standards. The reliability of above methods was compared by correlation coefficient based on observed and expected values.

RESULTSFor all three methods, measurement of the standards showed a linear correlation between the expected and detected values, i.e., PCR-RFLP (R(2)=0.828), RT-ARMS-qPCR (R(2)=0.998) and pyrosquencing (R(2)=0.997). For the MIDD family, it was consistent that there are 13 members carrying the A3243G mutation with different heteroplasmic levels. And there was no significant difference between the results by RT-ARMS-qPCR and pyrosquencing.

CONCLUSIONPCR-RFLP is not appropriate for the quantitative detection but could be used for early clinical screening. Both RT-ARMS-qPCR and pyrosquencing are suitable for the detection of low heteroplasmic level of A3243G mutation. Compared with pyrosquencing, RT-ARMS-qPCR is rapid, reliable and cost-effective, and is the best choice for detecting low mutation loads.

Adolescent ; Adult ; Base Sequence ; Child ; DNA, Mitochondrial ; genetics ; Deafness ; diagnosis ; genetics ; Diabetes Mellitus ; diagnosis ; genetics ; Female ; Genetic Testing ; methods ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Point Mutation ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Young Adult