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【Objective】 To establish RH gene mRNA sequencing method based on nanopores sequencing and to explore the RHD and RHCE mRNA transcripts in D positive and Del individuals. 【Methods】 From March 2021 to May 2022, 5 RhD positive samples and 5 Del samples screened out by hospitals in Chengdu were sent to our laboratory for futher examination. The erythrocytes and buff coat were isolated, then DNA and RNA were extracted.All 10 samples were genotyped by PCR-SSP. After the mRNA was reversely transcribed into cDNA, the full-length mRNA of RHD and RHCE genes were simultaneously amplified by a pair of primers. Sanger sequencing and third-generation sequencing technology based on Nanopore were used to sequence the amplified products, and the types and expressions of different splices of RHD and RHCE gene mRNA transcripts were analyzed. 【Results】 The method established in this study can simultaneously amplify the full length transcripts of RHD and RHCE. Ten different RHD gene mRNA transcripts and nine RHCE gene mRNA transcripts were detected in 10 samples. RHD full-length transcript (RHD-201) can be detected in RhD Del type, but the expression amount was significantly lower than that in RhD positive samples. The expression amount of transcript RHD-207 (Del789) in Del samples was significantly higher than that in RhD positive samples. The transcript RHD-208 (Del8910+ 213) was only detected in RhD Del type individuals, and no significant difference was found between other RHD transcripts and all RHCE transcripts in the two phenotypes. 【Conclusion】 In this study, an analytical method for sequencing full-length transcript isomers of RHD and RHCE mRNA via the third generation was successfully established, and complex alternative splicing patterns were found in RHD and RHCE genes, providing a new method for the study of alternative splicing of blood group gene variants mRNA.
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Objective:To investigate the regulatory effects of mitofusin 1 (MFN1) on lipopolysaccharide (LPS)-induced Raw264.7 mouse macrophages pyroptosis and to provide reference for further study on the prevention of inflammation and fibrosis caused by macrophage dysfunction.Methods:Raw264.7 mouse macrophages were cultured in vitro and used to construct a model of LPS-induced pyroptosis. CCK-8 staining, PI staining, LDH release assay and Western blot were used to verify the Raw264.7 pyroptosis induced by LPS. MFN1 expression was detected by Western blot. DCFH-DA probe was used to detect the synthesis of total reactive oxygen species (ROS); Mito-SOX was used to detect mitochondrial ROS; JC-1 mitochondrial membrane potential was detected by fluorescence probe to reflect mitochondrial damage. Based on Ubibrowser database, it was predicted that MFN1 could bind to a variety of E3 ubiquitin ligases. Then, immunofluorescence and co-immunoprecipitation (CO-IP) were used to analyze MFN1 ubiquitination. An overexpression plasmid for MFN1 was constructed and transfected into Raw264.7 cells to detect the changes in pyroptosis and mitochondrial function. Results:LPS could induce the pyroptosis of Raw264.7 cells and mitochondrial dysfunction. MFN1 expression was decreased after LPS stimulation. Ubiquitinated MFN1 was detected by CO-IP. Ubiquitination inhibitor MG-132 inhibited LPS-induced expression of pyroptosis-related proteins including NLRP3, Pro-caspase-1, Caspase-1, IL-1β and IL-18 and improved mitochondrial function. MFN1 overexpression relieved the mitochondrial dysfunction and pyroptosis of Raw264.7 cells induced by LPS.Conclusions:The ubiquitination of MFN1 induced by LPS was involved in mitochondrial dysfunction and macrophage pyroptosis, suggesting that MFN1 was a potential target for the treatment of macrophage-induced inflammation and related diseases.
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O 6-carboxymethyl guanine(O 6-CMG) is a highly mutagenic alkylation product of DNA that causes gastrointestinal cancer in organisms. Existing studies used mutant Mycobacterium smegmatis porin A (MspA) nanopore assisted by Phi29 DNA polymerase to localize it. Recently, machine learning technology has been widely used in the analysis of nanopore sequencing data. But the machine learning always need a large number of data labels that have brought extra work burden to researchers, which greatly affects its practicability. Accordingly, this paper proposes a nano-Unsupervised-Deep-Learning method (nano-UDL) based on an unsupervised clustering algorithm to identify methylation events in nanopore data automatically. Specially, nano-UDL first uses the deep AutoEncoder to extract features from the nanopore dataset and then applies the MeanShift clustering algorithm to classify data. Besides, nano-UDL can extract the optimal features for clustering by joint optimizing the clustering loss and reconstruction loss. Experimental results demonstrate that nano-UDL has relatively accurate recognition accuracy on the O 6-CMG dataset and can accurately identify all sequence segments containing O 6-CMG. In order to further verify the robustness of nano-UDL, hyperparameter sensitivity verification and ablation experiments were carried out in this paper. Using machine learning to analyze nanopore data can effectively reduce the additional cost of manual data analysis, which is significant for many biological studies, including genome sequencing.
Subject(s)
Deep Learning , Guanine , Nanopore Sequencing , Nanopores , Porins/geneticsABSTRACT
【Objective】 To investigate the risk factors of vasovagal reactions(VVR) related to plasma donation, so as to put forward clinical suggestions for early identification and accurate intervention of high-risk groups to ensure the safety of plasma donation. 【Methods】 The demographic characteristics(i.e. gender, age, weight) and records of plasma donors(donation history, pulse before plasma donation, duration of collection, etc.) were collected from July to December 2019 in a region of Sichuan. Based on logistic regression analysis, the correlation between these factors and the risk of VVR was explored. 【Results】 The information of 69 172 donors was collected, and the incidence of VVR was 7.04‰. The risk of VVR was reduced by 99% in the group with plasma collection duration less than 30 minutes compared with the group with plasma collection duration more than 50 minutes(OR, 0.01; 95% CI, 0.00~0.01; P<0.001). The risk of male group was 94 % lower than that of female group(OR, 0.06; 95% CI, 0.04~0.10; P<0.001). Compared with the 45~50 kg group, the risk of weight greater than 80 kg group decreased by 80%(OR, 0.20; 95% CI, 0.09~0.42; P<0.001). The risk of repeated donation group was 34 % lower than that of the first time donation group(OR, 0.66; 95% CI, 0.47~0.91; P<0.001). The risk of VVR in the group with pulse greater than 90 bpm before plasma donation was 2.43 times that in the 60~69 bmp group(OR, 2.43; 95% CI, 1.75~3.36; P<0.001). 【Conclusion】 Duration of plasma collection, gender, weight, frequency of plasma donation, pulse before plasma donation and donor status are independent risk factors for plasma donation-related VVR. Among them, plasma collection duration, gender and weight were the main independent risk factors for plasma donation-related VVR. For donors with plasma collection duration more than 50 minutes, female and low weight, higher risk of VVR was presented and more preventive intervention should be given.
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【Objective】 To analyze the genetic background of RhD-negative blood donors by detecting RHD and RHCE genes of those donors. 【Methods】 From March 2021 to May 2022, the blood samples of RhD-negative blood donors, who had been screened out by RhD primary screening and confirmatory experiments in the Yaan Blood Center, were firstly identified whether the RHD allele was completely deleted, then whether there were deletions in 10 exons of non-RHD allele complete deletion samples, finally, the remaining samples without RHD alleles and exon deletions were further analyzed by DNA sequencing. RHCE gene was detected by SSP-PCR method. 【Results】 Among the RHD gene test results of 104 RhD-negative samples, 65 cases were completely deleted (d/d), 33 were RHD partially deleted (one allele deletion), and 6 were without RHD gene deletion. The RHD alleles of 33 samples with partial deletion were detected by 10 exons, 13 had partial exon deletion, with genotype as RHD*D-CE(3-9)-D/d and phenotype as RhD negativity, and the remaining 20 samples had no exon deletion. The exon sequencing results of the non-deletion samples showed RHD*1227A/RHD*1227A in 6 samples, RHD*1227A/d in 19, RHD*3A/d in 1; both of the last two were considered Del by ISBT. The RHCE gene test results showed that all cc genotype blood donors were RhD true negative, while Del blood donors had no cc genotype. 【Conclusion】 Through the genetic background study of RhD negative blood donors, it is found that there is a high proportion of Del with weak expression of RhD antigen, whether this blood type affects clinical blood safety needs further researches.
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Through the screening of blood donors, detection of pathogen antigen and antibody, full coverage of nucleic acid detection, the risk of infectious blood transfusion has been reduced to a very low level. Especially, pathogen inactivation technology (PRT) has played an irreplaceable role in ensuring blood safety. The best way to inactivate pathogens is not only to effectively remove the target pathogens in the blood, but also to maintain the activity of active ingredients in the blood to the maximum extent, and it doesn′t affect the effect of blood therapy. In this paper, the development of pathogen inactivation technology is summarized, and the influence of pathogen inactivation treatment on the quality of blood components is discussed. It provides references for improving or developing new processing technology.
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Objective:To investigate and analyze the somatic symptom disorder, anxiety and depression in patients with myocardial bridge.Methods:A total of 276 patients with myocardial bridge diagnosed by coronary angiography (CAG) were enrolled in the department of cardiology of Renji hospital in Shanghai from June to December in 2016. There were 151 cases of simple myocardial bridge (no coronary stenosis or coronary artery stenosis <30%) and 125 cases of complex myocardial bridge (combined with coronary stenosis >30%). A total of 1067 patients with myocardial bridge without coronary angiography were collected at the same time. Self-rating somatic symptom scale (SSS), generalized anxiety disorder (GAD-7) and patient health questionnaire (PHQ -9) were given to these patients during hospitalization. At the same time, somatic symptoms disorder and anxiety and depression in the myocardial bridge group and non-myocardial bridge group were compared.Results:The prevalence of somatic symptom disorder in patients with myocardial bridge was higher than that in non-myocardial bridge patients (35.86% vs 28.30%, P<0.05). There was significant correlation between somatic symptom disorder and depression and anxiety, with correlation coefficients of 0.629 and 0.565, respectively. The prevalence of depression and anxiety in myocardial bridge patients was higher than that in non-myocardial bridge patients (depression: 23.91% vs 22.11%. P=0.467; anxiety: 17.02% vs 14.15%, P=0.22), but there was no statistical difference. For male patients or female patients, the prevalence of somatic symptom disorder, depression and anxiety in the simple myocardial bridge patients were higher than those in the complex myocardial bridge patients, but there was no statistical difference. The most common non-specific somatic symptoms disorder in patients with myocardial bridge were fatigue (64.5%), followed by sleep disorders (63.8%) and decreased attention (63.0%). Conclusion:The somatic symptom disorder in patients with myocardial bridge is significantly higher than that in non-myocardial bridge group. Especially for patients with myocardial bridge with non-specific somatic symptoms, early identification of somatic symptoms disorder of myocardial bridge patients will be beneficial to proper clinical invitation.
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To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.
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BACKGROUND:Studies have found that c-kit+ bone marrow mesenchymal stem cels can differentiate into myocardial cels specificaly, which may be the ideal seed cels. OBJECTIVE:To investigate the feasibility of cultivation of myocardial tissue by using c-kit+ bone marrow mesenchymal stem cels and decelularized heart matrix. METHODS:Heart tissues harvested from adult rats were decelularized for the folowing experiments. Primary rat bone marrow-derived mesenchymal stem cels were culturedin vitro. Until passage 8, bone marrow mesenchymal stem cels were enriched for c-kit and induced by 5 μmol/L 5-azacytidine for 2 weeks, and a second enrichment for the dihydropyridine receptor subunit α2δ1 was performed before analysis of cardiac differentiation or implantation into decelularized heart matrix for cultivation of myocardial tissue. Six weeks later, myocardial differentiation was identified by specific cardiac protein and action potential. Immunofluorescence staining was used to analysis neonatal myocardial tissue. RESULTS AND CONCLUSION:Six weeks after the second enrichment, 60% bone marrow mesenchymal stem cels expressed cardiac troponin T, GATA binding protein 4, and connexin 43, and these cels could be induced to yield cardiac action potential, which was identified as cardiac differentiation. And when implanted into decelularized heart matrix, these cels could form myocardial tissue arranged regularly.