[ABO gene subtypes and gene expression analysis in three cases of hematological malignancies patients].

Objective: To explore the application and discovery of genotyping, gene sequencing, and gene expression analysis in the determination of ABO blood group subtypes and antigen expression abnormalities in hematological malignancies patients. Methods: From June 2019 to May 2020, three clinical cases were found with forward and reverse ABO typing discrepancy or atypical serologic agglutination pattern in the laboratory and blood transfusion department of Hebei Yanda Ludaopei Hospital were selected. Sequence-specific primer PCR (PCR-SSP) and Sanger sequencing of ABO gene coding regions were performed to determine the ABO genotypes, and whole transcriptome sequencing was used to analyze ABO and FUT1 gene expression levels. Results: A 12-year-old female acute lymphoblastic leukemia patient was determined as O.01.02 and BA.04 sub-genotype, corresponding to the serological B(A) subtype, and her ABO gene expression was normal (354.80). A 41-year-old female acute myeloid leukemia patient was determined as A1.02 and B.01 genotype, corresponding to the serological A(1)B phenotype, and her ABO gene expression was significantly reduced (45.70). A 42-year-old male with myelodysplastic syndrome and myelofibrosis was determined as A1.02 and A2.05 sub-genotype, corresponding to the serological A(1) and A(2) phenotype, respectively, and his ABO expression was negative. FUT1 expression was in the normal range in all three cases. The clinical blood product infusion strategy was formulated according to the genotype and the corresponding immunological subtype, and no significant transfusion-related adverse reactions occurred. Conclusion: Blood group sub-genotypes or aberrant gene expression can lead to ambiguities in serological blood group determination in hematological malignancies patients. ABO genotyping and gene expression analysis can help in this scenario and escort blood product infusion safety.

[Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay].

Objective: To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine. Methods: HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID(50), respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine. Results: The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 µg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 µg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×10(4) and 1×10(5). The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05). Conclusion: We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Transcriptome network-based method to identify genes associated with unruptured intracranial aneurysms.

Intracranial aneurysm is a balloon or sac-like dilatation of blood vessels inside the brain. Despite their importance, the biological mechanisms of intracranial aneurysms are not totally understood. We used public genome-wide gene expression profile data to identify potential genes that are involved in intracranial aneurysm in order to construct a regulation network. Some of the transcription factors and target genes that we identified in this network had been identified as related to intracranial aneurysm in previous studies. We found additional transcription factors and target genes that are apparently related to intracranial aneurysm with this method. The confirmation of previously identified genes and transcription factors supports the usefulness of this transcriptome network analysis for the identification of candidate genes involved in intracranial aneurysm.

Fatty acid positional specificity in phospholipids of L1210 leukemia and normal mouse lymphocytes.

The positional distribution of fatty acids in the choline and ethanolamine phosphoglycerides of the L1210 murine leukemia cells was determined and compared to that of normal mouse lymphocytes. The major phospholipids of both cell types had appreciable degrees of positional specificity as evident from the higher percentage of saturated fatty acids in position 1 and of polyunsaturated fatty acids in position 2. The L1210 cells had less arachidonate and more linoleate in position 2 of choline and ethanolamine phosphoglycerides as compared to the normal lymphocytes. However, there were similar proportions of saturated, monoenoic and polyenoic fatty acids in positions 1 and 2 of the phospholipids of the L1210 leukemia cells and the lymphocytes, These data demonstrate that fatty acid positional specificity is retained in the major phospholipids of this rapidly growing tumor.

Fatty acid metabolism in L1210 murine leukemia cells: differences in modification of fatty acids incorporated into various lipids.

L1210 leukemia cells can utilize all of the main fatty acids that normally are present in the ascites fluid in which they grow. This finding is consistent with the view that L1210 cells derive most of their fatty acids from the ascites fluid. From 80--90% of each fatty acid was incorporated into cell lipids without structural modification, suggesting that the lipid composition of these cells can be altered by changing the type of fatty acids to which they are exposed. Most importantly, the palmitate that was subsequently incorporated into total cell phospholipids was elongated and desaturated somewhat more than that incorporated into triglycerides. This difference was due primarily to more extensive modification of the palmitate incorporated into the ethanolamine phosphoglycerides fraction. Although there was no difference between total phospholipids and triglycerides with linoleate, more of the linoleate incorporated into ethanolamine phosphoglycerides was elongated and further desaturated than that incorporated into choline phosphoglycerides and triglycerides. These findings indicate fatty acids incorporated into various cell lipid fractions are not structurally modified to the same extent. There appears to be greater modification of fatty acid used for ethanolamine phosphoglyceride synthesis as compared with triglyceride and choline phosphoglyceride synthesis.

Fatty acid utilization by L1210 murine leukemia cells.

L1210 murine leukemia cells grow in an ascites plasma that contains lipids, including 0.62 +/- 0.046 (S.E.) MICRONEq free fatty acid per ml. in vitro incubations demonstrated that isolated L1210 cells readily utilize free fatty acid that is added to the incubation medium. When the cells were incubated with albumin-bound [1-14C]palmitate, about 12 times more radioactivity was incorporated into cell lipids than was oxidized to CO2. Triacylglycerols contained 1.5 to 4 times more radioactivity than phospholipids, and from 48 to 69% of the phospholipid radioactivity was recovered in the choline phosphoglycerides. [1-14C]Palmitate utilization increased as the fatty acid concentration of the medium was raised, the largest increase occurring in the triacylglycerol fraction. Palmitate utilization also was increased by the presence of carbohydrates in the medium, their effectiveness (in descending order) being glucose, mannose, galactose, fructose, and glycerol. By contrast, ribose did not produce any stimulatory effect. During a 1-hr incubation, between 82 and 87% of the [1-14C]palmitate that was taken up remained as palmitic acid. From 8 to 15% was elongated to stearate, and only 2 to 3% was desaturated to palmitoleate and oleate. Based upon the lipid content, growth rate, and palmitate utilization rate of the cells, it appears that a major portion of the lipid requirements of the L1210 cell may be supplied by the fatty acid contained in the ascites plasma. In addition, our results suggest that most of the saturated fatty acid taken up is incorporated into cell lipids without structural modification.