RNA-seq can help us quickly obtain the whole transcriptome sequences of species under different conditions. Many Unigenes that are assembled by raw reads always do not contain complete open reading frame (ORF). In addition, it also has some redundancy in transcriptome library. Some Unigenes in the library, although belong to one transcript, cannot be assembled without overlapping. We found five incomplete Unigenes annotated ammonium transporter (AMT) from Salicornia europaea transcriptome, in which two Unigenes (Uni4 and Uni5) had identical expression patterns across four transcriptomes. The two Unigenes may come from one transcript. Analyzing the Unigene position of transcript by NCBI blastx, we found that Uni4 and Uni5 respectively located in 5' end and 3' end compared with the reference transcript, and an unknown gap of 120 bp may exist in a hypothetic transcript to which Uni4 and Uni5 both belong. To verify the hypothesis, single forward primer and single reverse primers were respectively designed on Uni4 and Uni5, and a fragment with about 800 bp was generated by PCR. Then it was sequenced and aligned with Uni4 and Uni5. Finally, we assembled a sequence with 1 667 bp, which contains a complete ORF (1 482 bp, coding 494 amino acids). It belongs to amt1 subfamily and was named Seamt1 via the phylogenetic analysis. It was pointed by bioinformatics tools that SeAMT1 protein conformed to the AMT characteristics of other species. This work clustered expression pattern to explore the Unigenes of one transcript, and the feasibility of this method was validated through the other two groups of Unigenes. The handy method will benefit extension and assembling of Unigene in transcriptome, it also helps achieve the complete ORF and gene function.
Ammonium Compounds ; Chenopodiaceae ; genetics ; Computational Biology ; Gene Expression Profiling ; Genes, Plant ; Membrane Transport Proteins ; genetics ; Molecular Sequence Annotation ; Open Reading Frames ; Phylogeny ; Plant Proteins ; genetics ; Transcriptome

Objective To investigate the biological effect of anti-leukemic cells induced by eosinophilic granulocyte (EOS) in bone marrow of patients with chronic myelogenous leukemia (CML).Methods The BCR-ABL fusion gene as well as the expression of IL-12 and IL-17 mRNA were performed by RT-PCR.The serum concentrations of cytokine IL-12 and IL-17 were determined by enzyme-linked immuno sorbent assay (ELISA).Immunochemistry staining and cytochemistry staining were used to observe the peroxydase (POX) and human Leukocyte antigen (HLA)-DR expression of EOS in bone marrow.Immunofluorescence staining was used to observe mannose receptor (MR),IL-12,IL-17A and IL-17receptor A (IL-17RA) expression of EOS.The results between the CML patients and the healthy controls were compared.Results Serum levels of IL-12 and IL-17 were higher in the 60 CML patients [(196.33 ±21.79) ng/L and (36.55-±3.01) ng/L] than those in the controls [(96.60 ±4.92) ng/L and (23.74 ±1.36) ng/L].In the 32 patients with activated EOS,the levels of IL-12 and IL-17 were (273.12 ± 17.16)ng/L and (40.11 ± 6.13) ng/L,which were significantly higher than those in the non-activated EOS [(126.16 ± 14.27) ng/L and (28.14 ±5.29) ng/L] (P values ＜0.01).IL-12 and IL-17 mRNA were expressed in activated EOS,while BCR-ABL fusion gene was not found.The amounts of EOS were increased abnormally in the bone marrow and peripheral blood of the CML patients with POX positive staining in the cytoplasm and weakly positive HLA-DR staining.It was observed easily by a microscope that EOS could attack leukemic cells in bone marrow through adhesion,capture and phagocytosis.Activated EOS could express IL-12,IL-17A and MR,which was related with the serum levels of these cytokines.Conclusions Activated EOS in bone marrow of CML patients could express IL-12 and IL-17.Activated EOS could induce coup injury to leukemic cell by releasing POX and expressing IL-12 and IL-17.It can also capture or swallow target cells via the expression of MR on the membrane.EOS may play an important role in the anti-tumor immunologic function in bone marrow of CML patients.

Objective To investigate the changes of platelet reactivity and its influencing factors after aspirin treatment in patients with ischemic stroke complicated with diabetes. Methods From September 2016to December 2017, patients with acute ischemic stroke admitted to the Department of Neurology, Weihai Municipal Hospital within 24 h of onset were enrolled. All patients took aspirin (100 mg/d) within 24 h ofadmission, and after taking the drug (7 ±2 d), the PL-11 platelet function analyzer was used to determine the maximum platelet aggregation ratio (MAR) induced by arachidonic acid (AA). The baseline data of the patients were documented. The factors affecting high platelet reactivity (HPR) were analyzed. Results A total of 398 patients with ischemic stroke were enrolled, including 137 in the diabetes group and 261 in the non-diabetes group. MARAA (43. 45％ ± 14. 11％ vs. 31. 55％ ± 19. 39％; t = 6. 996, P < 0. 001) and the incidence of HPR (34. 3％ vs. 19. 9％; χ2 = 9. 946, P = 0. 002) in the diabetes group were significantly higher than in those in the non-diabetes group. Of the 137 patients with ischemic stroke complicated with diabetes, 47 had HPR. The proportions of patients with hyperlipidemia, previous history of stroke or transient ischemic attack and baseline NIHSS score, HOMA-IR (homeostatis model assessment-insulin resistance),high-sensitivity C-reactive protein, fasting blood glucose, and glycosylated hemoglobin in the HPR group were significantly higher than those in the non-HPR group (all P < 0. 05). Multivariate logistic regression analysis showed that HOMA-IR (odds ratio [OR] 1. 153, 95％ confidence interval [CI] 1. 027-1. 295; P =0. 016), high-sensitivity C-reactive protein (OR 9. 416, 95％ CI 2. 271-39. 049; P = 0. 002), fasting blood glucose (OR 1. 125, 95％ CI 1. 025-1. 235; P = 0. 013), and glycosylated hemoglobin (OR 1. 458, 95％ CI 1. 170-1. 816; P = 0. 001) were the independent risk factors for HPR. Conclusion The platelet reactivity during aspirin therapy in patients with ischemic stroke complicated with diabetes mellitus was high, and platelet activity was associated with multiple mechanisms, such as inflammation, insulin resistance, and hyperglycemia.

Objective To research the influence of hypoxic preconditioning in anti-oxidative ability of brain tissues and neurological functions in rats after traumatic brain injury.Methods Forty eight adult male Sprague Dawley rats were randomly divided into sham-operated group,hypoxic preconditioning group (HPC),traumatic brain injury group (TBI) and hypoxic preconditioning+traumatic brain injury group (HPCT,n=12).The HPC rat models were made by hypobaric chamber for 3 d (50 kPa,3 h/d) and TBI were induced by Feeney's improved equipment.All rats were killed 24 h after injury,and the neurological functions of each group were evaluated by modified neurologieal severity scale (mNSS);the neuronal survival around contusion area was detected by NeuN immunohistochemical staining,and the protein and mRNA expressions of nuclear transcription factor-related factor 2 (Nrf2) and thioredoxin reductases 2 (TrxR2) in the brain tissues were detected by Western blotting and real-time quantitative PCR.Results The mNSS scores in the TBI and HPCT groups were significantly higher as compared with those in the sham-operated and HPC groups (P＜0.05),but those in the HPCT group was significantly lower than those in the TBI group (P＜0.05).The neuronal survival in the TBI and HPCT groups was decreased as compared with that in the sham-operated and HPC groups (NeuN expressions:0.274±0.033,0.281±0.042 vs 0.124±0.014,0.150±0.019),with significant difference (P＜0.05),while that in the HPCT group was statistically increased as compared with that in the TBI group (P＜0.05).The expressions of Nrf2 and TrxR2 in the brain tissues of TBI and HPCT groups were significantly increased as compared with those in the sham-operated and HPC groups (P＜0.05),and those in the HPCT group was significantly up-regulated as compared with those in the TBI group (P＜0.05).Conclusion HPC can increase the anti-oxidative ability and relieve the neurological functions missing after TBI.

Objective To investigate the expression of Toll-like receptor 2(TLR2)and Toll-like receptor 4(TLR4)in peripheral blood mononuclear cells(PBMC)in children with recurrent respiratory tract infection(RRTI)and its relationship with T helper cell 1(Th1)/T helper cell 2(Th2)immune response.Methods A total of 65 children diagnosed with RRTI who admitted to the hospital from December 2020 to December 2022 were enrolled in the study as the RRTI group,and 45 healthy children who underwent physical examination in the hospital during the same period were enrolled as the control group.The relative expression levels of TLR2 and TLR4 mRNA in PBMCs were detected by real-time fluorescence quantitative PCR(qPCR).The expres-sion rates of TLR2 and TLR4 protein in PBMCs were detected by flow cytometry.The levels of Th1 cytokine interferon-γ(IFN-γ),Th2 cytokine interleukin-4(IL-4)and their ratio(IFN-γ/IL-4)in plasma were detected by enzyme-linked immunosorbent assay(ELISA).Pearson correlation analysis was used to analyze the corre-lation between TLR2,TLR4 protein expression rates and plasma IFN-γ,IL-4 levels.Results The RRTI group had significantly higher plasma level of Th2 cytokine IL-4 than the control group,significantly lower plasma level of Th1 cytokine IFN-y than the control group,and significantly lower ratio of IFN-γ/IL-4 than the con-trol group,the differences were all statistically significant(P＜0.05).The relative expression levels of TLR2 and TLR4 mRNA and protein expression rates in PBMC of children in the RRTI group were higher than those in the control group,and the differences were statistically significant(P＜0.05).Pearson correlation analysis showed that the protein expression rates of TLR2 and TLR4 in PBMC of children with RRTI were both nega-tively correlated with both plasma IFN-γ levels and IFN-γ/IL-4(P＜0.05)and positively correlated with plasma IL-4 levels(P＜0.05).Conclusion The expression of TLR2 and TLR4 in PBMC and plasma Th1/Th2 cytokines in children with RRTI may be involved in the occurrence and development of the disease.Ex-cessive activation of TLR2 and TLR4 may weaken Th1 function and enhance Th2 function.

Objective:To explore the predictive value of thrombus markers for venous thromboembolism (VTE) in patients with malignant tumors after surgery.Methods:The clinical data of 150 patients with malignant tumors after surgery admitted to Cangzhou Hospital of Integrated Traditional Chinese Medicine and Western Medicine in Hebei Province from July 2020 to February 2021 were retrospectively analyzed. All 150 patients followed-up for 4 weeks were treated as the observation group, including 30 cases with VTE (the thrombosis group) and 120 cases without VTE (the non-thrombosis group). Another 60 cases undergoing healthy physical examination during the same period were selected as the control group. The chemiluminescence immunoassay was used to detect thrombin-antithrombin complex (TAT), plasmin-α 2 plasmin inhibitor complex (PIC), thrombomodulin (TM), tissue-type plasminogen activator inhibitor-1 complex(tPAIC). The control group was tested once, and the observation group was tested on the 1 day before the operation and 1 day after the operation. Receiver operating characteristic (ROC) curve was used to evaluate the predictive value of thrombus markers for VTE in patients with malignant tumors after surgery. Results:The patients with lung cancer ( χ2 = 12.53, P = 0.014), ≥ 60 years old ( χ2 = 6.66, P = 0.036),body mass index>30 kg/m 2 ( χ2 = 40.53, P<0.001), tumor metastasis ( χ2 = 5.38, P = 0.031), Ⅲ-Ⅳ stage ( χ2 = 5.83, P = 0.023) had higher incidence of VTE after the operation, and the difference was statistically significant. The levels of TAT, PIC and TM in the observation group were higher than those in the control group (all P < 0.05).The levels of TAT and TM in the thrombosis group were higher than those in the non-thrombosis group before the operation, and the difference was statistically significant (all P < 0.05).The value of TM in predicting VTE was high [the best cut-off value was 10.70 TU/ml, area under the curve (AUC) was 0.786, the sensitivity was 73.30%, the specificity was 81.70%], the combination of TAT and TM could improve the predictive value (AUC was 0.796, the sensitivity was 80.00%, the specificity was 77.50%). The levels of TAT, PIC, TM and tPAIC in thrombosis group were all higher than those in the non-thrombosis group after the operation, and the difference was statistically significant (all P < 0.05). The value of TAT in predicting VTE was high (the best cut-off value was 16.50 ng/ml, AUC was 0.887, the sensitivity was 82.36%, the specificity was 71.65%), the combination of TAT, PIC, TM and tPAIC could improve the predictive value (AUC was 0.913, the sensitivity was 90.00%, the specificity was 88.60%). The level of PIC was positively correlated with TAT before and after the operation ( r = 0.66, P < 0.001; r = 0.64, P < 0.001). Conclusions:TM can be used as a sensitive indicator in the early prediction of VTE for the patients with malignant tumors and it aims at the prevention; TAT can be used as a specific indicator in predicting the development of VTE and it aims at the intervention in time. The combined detection of TAT, PIC, TM and tPAIC can improve the predictive value of VTE. At the same time, PIC can be used to evaluate the risk of bleeding.

RNA-binding protein exerts important biological function by specifically recognizing RNA motif. SELEX (Systematic evolution of ligands by exponential enrichment), an in vitro selection method, can obtain consensus motif with high-affinity and specificity for many target molecules from DNA or RNA libraries. Here, we combined SELEX with next-generation sequencing to study the protein-RNA interaction in vitro. A pool of RNAs with 20 bp random sequences were transcribed by T7 promoter, and target protein was inserted into plasmid containing SBP-tag, which can be captured by streptavidin beads. Through only one cycle, the specific RNA motif can be obtained, which dramatically improved the selection efficiency. Using this method, we found that human hnRNP A1 RRMs domain (UP1 domain) bound RNA motifs containing AGG and AG sequences. The EMSA experiment indicated that hnRNP A1 RRMs could bind the obtained RNA motif. Taken together, this method provides a rapid and effective method to study the RNA binding specificity of proteins.
Aptamers, Nucleotide ; Gene Library ; Heterogeneous Nuclear Ribonucleoprotein A1 ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; RNA ; SELEX Aptamer Technique

Protein-RNA interaction networks are essential to understand gene regulation control. Identifying binding sites of RNA-binding proteins (RBPs) by the UV-crosslinking and immunoprecipitation (CLIP) represents one of the most powerful methods to map protein-RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method (GoldCLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein (PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. GoldCLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.
Binding Sites ; Cell Line ; Heterogeneous-Nuclear Ribonucleoproteins ; isolation & purification ; metabolism ; Humans ; Immunoprecipitation ; methods ; Polypyrimidine Tract-Binding Protein ; isolation & purification ; metabolism ; RNA ; isolation & purification ; metabolism ; RNA-Binding Proteins ; isolation & purification ; metabolism