1. Fatty acid quantification method by isobutyl esterification and GC-FID/MS
Journal of Shanghai Jiaotong University(Medical Science) 2020;40(1):22-29
Objective:To establish a quantification method for understanding the varieties and concentrational changes of fatty acids under physiological and pathophysiological conditions. Methods:Based on isobutyl esterification using (-)-menthyl chloroformate and isobutanol, 27 typical fatty acids were qualitatively and quantitatively analyzed by gas chromatography-flame ionization detector/mass spectrometry (GC-FID/MS). The sensitivity and stability of the method were detected by limit of detection (LOD), limit of quantification (LOQ), and intra-day and inter-day relative standard deviation (RSD). The method was applied to four typical biological samples (human serum, urine, feces and rat liver) to verify the universality in different substrates. Results:Isobutyl esters of 27 fatty acids were effectively separated with an HP-5MS column. The results showed nice linearity within 2-3 orders of magnitude in terms of concentration with R2>0.99 for all 27 tested fatty acids. The LODs of the method were between 0.03 and 2.96 pmol on column whereas the LOQs were between 0.09 and 9.86 pmol. The results of method validation showed that the intra-day and inter-day RSDs were all under 10% in the range of high, medium and low concentrations. With this method, moreover, 10, 7, 14 and 9 fatty acids were detected in human serum, urine, feces and rat liver, respectively. Conclusion:A quantitative analytical method for fatty acids with short, media and long chains (C1-C24) is established based on isobutyl estrification in aqueous media. This method has the advantages of mild condition, high sensitivity, good stability, and simple and quick operation, and can be directly used in the detection of serum, urine, feces, liver and other biological samples, which may provide a new idea for high-throughput metabolome analysis.
2.Human nasal type NK/T cell lymphoma: study of differential expression genes by cDNA microarray.
Miao-xia HE ; Wei-ping LIU ; Gan-di LI ; Fan YANG ; Wen-yan ZHANG ; Sha ZHAO ; Qiong-lan TANG
Chinese Journal of Pathology 2005;34(7):426-427
Gene Expression Profiling
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Humans
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Killer Cells, Natural
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pathology
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Lymphoma, T-Cell
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genetics
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metabolism
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pathology
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Nose Neoplasms
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genetics
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metabolism
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pathology
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Oligonucleotide Array Sequence Analysis
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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biosynthesis
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genetics
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RNA, Messenger
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biosynthesis
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genetics
3.HBV-induced metabolic changes in HepG2 human hepatoma cells revealed by gas chromatography-mass spectrometry
chen Jing LI ; fen Qian WAN ; yan Miao GAN ; ru Hui TANG
Journal of Shanghai Jiaotong University(Medical Science) 2017;37(10):1322-1326
Objective · To investigate the impacts of hepatitis B virus (HBV) infection on the metabolomic phenotype of HepG2 human hepatoma cells.Methods · With gas chromatography-mass spectrometry (GC-MS), metabolite composition of HepG2 and HepG2.2.15 cells (derived from HepG2 cells transfected with a plasmid containing HBV) were analysed. Results · GC-MS analysis mainly found 34 metabolites in both HepG2 and HepG2.2.15 cells,including glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), serine (Ser), threonine (Thr), methionine (Met), cysteine (Cys), cystine, aspartic acid (Asp), glutamic acid (Glu), pyroglutamic acid, phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), hypoxanthine, uracil,myo-inositol, lactic acid, succinic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, urea, cholesterol, etc. These metabolites were involved in multiple metabolic pathways including glycolysis and metabolism of fatty acids, amino acids, purines and pyrimidines. Compared with HepG2 cells,HepG2.2.15 cells had significantly higher levels in lactic acid, linolenic acid, Ala and Cys, but lower levels in Leu, Ile, Val, Phe, Met, Trp, Pro, Tyr, myoinositol and uracil. Conclusion · HBV infection dysregulates the metabolism of amino acids and fatty acids in hepatocytes. GC-MS analysis provides complimentary information about HBV-induced metabolic changes of host cells.
4.Expression of CYR61 and VEGF in extranodal nasal-type NK/T cell lymphoma and their significances.
Qiong-lan TANG ; Wei-ping LIU ; Wen-yan ZHANG ; Fan YANG ; Miao-xia HE ; Qing-jie XIA ; Gan-di LI
Chinese Journal of Hematology 2006;27(10):661-665
OBJECTIVETo investigate the expression of CYR61 and VEGF in extranodal nasal-type NK/T cell lymphoma and its significance.
METHODSCYR61 mRNA and VEGF mRNA were detected by real-time fluorescence quantitative PCR method in 20 cases of extranodal nasal-type NK/T cell lymphoma. Expressions of CYR61 and VEGF were studied by immunohistochemistry in 40 cases of the tumor.
RESULTS(1) Over-expression of CYR61 mRNA and VEGF mRNA was found in 19/20(95.0% ) and 15/20(75.0% ) cases, respectively. (2)Tumor cells expressing CYR61 protein and VEGF protein were detected in 38(95.0% ) and 25 (62. 5% ) of the 40 cases respectively, being no significant difference from the control. Co-expression of CYR61 and VEGF at both the mRNA and protein levels was 95.0% and 65.0% , respectively. Over-expression of CYR61 and VEGF at both mRNA and protein levels was found in 8 of the 40 cases. (3) The prognosis of the patients over-expressing CYR61 and VEGF was worse.
CONCLUSIONIn extranodal nasal-type NK/T cell lymphoma, the expression level of CYR61 and VEGF was changed and it may be of prognostic implication of
Adolescent ; Adult ; Aged ; Cysteine-Rich Protein 61 ; Female ; Humans ; Immediate-Early Proteins ; biosynthesis ; genetics ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Killer Cells, Natural ; Lymphoma, T-Cell ; metabolism ; pathology ; Male ; Middle Aged ; Nose Neoplasms ; metabolism ; pathology ; Polymerase Chain Reaction ; RNA, Messenger ; biosynthesis ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics
5.Mutational analysis of 3 Glanzmann's thrombasthenia pedigrees
Fangyan GAN ; Linzi MIAO ; Chenxue QU ; Yan GONG ; Yao LU ; Ran YOU ; Bingjing GAO ; Tao LI ; Shuai GUO
Chinese Journal of Laboratory Medicine 2019;42(4):262-269
Objective To explore the molecular pathogenesis of 3 Glanzmann's thrombasthenia pedigree by using bioinformatics software and provide evidence for in vitro experiments. Methods The genetic analysis of 3 pedigree diagnosed as Glanzmann's thrombasthenia was carried out. Clustalx-2.1 win software was used to analyze the conservatism of mutant sites in homologous sequences. Bioinformatics software such as PolyPhen-2, PROVEAN, SIFT and Mutationtaster was used to analyze the biological effect of mutation. SPDBV software constructed the molecular structure model of mutant protein and evaluated the influence of mutation on protein structure. Results The "new mutations" found in 3 Glanzmann's thrombasthenia pedigree were ITGA2B:c. 814G>C (p. Val272Leu), ITGA2B:c. 432G>A (p. Trp144Ter) and ACTN1:c. 2458A>G (p. Ile820Val). All three mutations were highly conserved among homologous species. Mutationtaster software showed that 3 new mutations were likely pathogenic. PolyPhen-2 and PROVEAN software showed ITGA2B p.Val272Leu and ACTN1 p.Ile820Val were benign and SIFT software showed that ITGA2B p. Val272Leu were likely pathogenic, while ACTN1 p. Ile820Val is benign. The result of SPDBV software showed that the Val272 of ITGA2B was transformed to Leu, neutralizing all the original hydrogen bond. The Trp144 of ITGA2B is transformed to Ter, resulting in the truncated proteins with only 113 amino acid residues. All these mutations affected the molecular structure of GPⅡb, resulting in a decrease ofGPⅡb/Ⅲa expression. When the Ile820 of ACTN1 is transformed to Val, onlyretained the hydrogen bond of Ile820 and Asp822, neutralized the rest hydrogen bond, whichaffected the molecular structure and protein function of ACTN1. Conclusion The mutations of ITGA2B:c.814G>C (p.VAL272LEU), ITGA2B:c.432G>A (p.Trp144Ter) and ACTN1:c.2458A>G (p.Ile820Val) are pathogenic.
6.Simulated microgravity conditions and carbon ion irradiation induce spermatogenic cell apoptosis and sperm DNA damage.
Hong Yan LI ; ; Hong ZHANG ; Guo Ying MIAO ; Yi XIE ; Chao SUN ; Cui Xia DI ; Yang LIU ; Yuan Yuan LIU ; Xin ZHANG ; Xiao Fei MA ; Shuai XU ; Lu GAN ; Xin ZHOU
Biomedical and Environmental Sciences 2013;26(9):726-734
OBJECTIVETo investigate the effect of simulated microgravity and carbon ion irradiation (CIR) on spermatogenic cell apoptosis and sperm DNA damage to the testis of male Swiss Webster mice, and assess the risk associated with space environment.
METHODSSperm DNA damage indicated by DNA fragmentation index (DFI) and high DNA stainability (HDS) was measured by sperm chromatin structure assay (SCSA). Apoptosis of spermatogenic cells was detected by annexin V-propidium iodide assay. Bax (the expression levels of p53) and proliferating cell nuclear antigen (PCNA) were measured by immunoblotting; p53 and PCNA were located by immunohistology.
RESULTSHDS, DFI, apoptosis index, and the expression levels of p53 and Bax were detected to be significantly higher in the experimental groups (P<0.05) compared with those in the control group; however, the PCNA expression varied to a certain degree. p53- and PCNA- positive expression were detected in each group, mainly in relation to the spermatogonic cells and spermatocytes.
CONCLUSIONThe findings of the present study demonstrated that simulated microgravity and CIR can induce spermatogenic cell apoptosis and sperm DNA damage. Sperm DNA damage may be one of the underlying mechanisms behind male fertility decline under space environment. These findings may provide a scientific basis for protecting astronauts and space traveler's health and safety.
Animals ; Apoptosis ; radiation effects ; Carbon ; Cell Proliferation ; radiation effects ; DNA Damage ; Heavy Ions ; adverse effects ; Immunohistochemistry ; Male ; Mice ; Random Allocation ; Sperm Count ; Spermatogenesis ; radiation effects ; Spermatozoa ; radiation effects ; Testis ; radiation effects ; Weightlessness Simulation
7.Prognostic and Immunological Role of ABHD5: a Pan-Cancer Analysis
Yu⁃tao CHEN ; Yang YANG ; Gan XIA ; Miao-hong XU ; Ji-zhao CAO ; Jia-yin LU ; Yan ZOU ; Xia YANG
Journal of Sun Yat-sen University(Medical Sciences) 2022;43(3):400-411
ObjectiveTo evaluate the ABHD5 expression in pan-cancer and its correlations with the main clinical stages, prognosis and immune cell infiltration. MethodsGTEx, TCGA, TIMER2.0, CPTAC databases, immunohistochemistry and western blot were used to analyze the expression levels of ABHD5 in different cancer tissues and adjacent tissues as well as correlations between ABHD5 expression and the main clinical stages. Kaplan-Meier Plotter, Oncolnc and R2 databases were used to explore the prognostic value of ABHD5. The relationship between ABHD5 and immune cell infiltration was analyzed by TCGA and TIMER2.0 databases. STRING and GEPIA databases were used to detect ABHD5-binding proteins and co-expression genes, which were then analyzed by GO and KEGG. ResultsThe mRNA and protein expression levels of ABHD5 were lower in cancer tissues than those in normal tissues in multiple cancer types, but higher in few cancer types. High-level expression of ABHD5 was related to better prognosis in 8 cancer types and related to worse prognosis in 2. ABHD5 expression levels were positively correlated with immune cell infiltration in 9 cancer types, and were the same with neutrophil infiltration and expression of immune checkpoints in pan-cancer. Enrichment analysis showed that ABHD5 was related to histone demethylation. ConclusionPossibly used as a potential prognostic predictor in pan-cancer, ABHD5 was also correlated with neutrophil infiltration, expression of immune checkpoints and histone demethylation.