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BACKGROUND:In consideration of the food safety and ecological safety of transgenic plants,the retention of marker genes is the primary safety issue affecting transgenic plants. OBJECTIVE:Based on the principle of immune caries prevention,our research team successfully constructed the plant anti-caries vaccine fusion gene expression vector pCAMBIA-E8-APB-DOCK8 for these two caries causing virulence factors surface protein and glucosyltransferase,which provides a basis for the research and development of transgenic plant vaccine. METHODS:In this study,the selective marker genes Km and GUS in the plant caries vaccine fusion gene expression vector pCAMBIA-E8-APB-DOCK8 were removed by DNA recombination technology through a series of steps such as DNA fragment separation,connection,transformation,clone detection,and sequencing. RESULTS AND CONCLUSION:The efficiency of marker gene removal was 99%.This study has laid a good experimental foundation for the safe production of transgenic plant vaccine against dental caries,and also provided ideas for the construction of other plant vaccine vectors.
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BACKGROUND:The expression efficiency of recombinant adeno-associated virus serotype 9(rAAV9)carrying the macrophage-specific promoter synthetic promoter 146-C1(SP146-C1)and the exogenous gene vascular endothelial growth factor C(VEGFC)in atherosclerosis is uncertain. OBJECTIVE:To investigate the expression efficiency of rAAV9-SP146-C1-VEGFC in atherosclerotic mice and its effect on lymphangiogenesis. METHODS:Thirty ApoE-/-mice were fed high-fat diet for 12 weeks to establish atherosclerosis models and were randomly divided into six groups,five in each group:7-,14-,21-,28-,and 35-day transfection groups and control group.The mice in the transfection groups were transfected with 5.0×1011 vg rAAV9-SP146-C1-VEGFC by caudal vein injection.In the control group,the mice were injected with the same amount of control virus rAAV9-SP146-C1-Scramble.Animals in the first five groups were killed under anesthesia at 7,14,21,28 and 35 days after transfection,respectively,and those in the control group were killed under anesthesia at 7 days.Serum,femur,tibia,heart and aorta tissue samples were collected and retained in each group.The femur and tibia of mice in each group were used to extract bone marrow-derived macrophages.The gene expression of vascular endothelial growth factor C(VEGFC),vascular endothelial growth factor receptor 3(VEGFR3),Podoplanin and lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1)in bone marrow-derived macrophages and the aorta were detected by RT-qPCR.VEGFC protein expression levels in bone marrow-derived macrophages and the aorta were detected by western blot,serum level of VEGFC was detected by ELISA,and VEGFC expression in the aortic sinus and LYVE-1 expression around the aorta and in the myocardium was detected by immunofluorescence. RESULTS AND CONCLUSION:The serum level of VEGFC,the mRNA expression of VEGFC,VEGFR3,Podoplanin,and LYVE-1 in bone marrow-derived macrophages and the aorta,the protein expression of VEGFC in bone marrow-derived macrophages,and the fluorescence intensity of VEGFC in aortic sinus plaques were significantly increased in the 7-day transfection group compared with the control group(P＜0.05,P＜0.01).Serum VEGFC level of mice transfected with rAAV9-SP146-C1-VEGFC gradually increased with time and began to decrease at 28 days.mRNA levels of VEGFC,VEGFR3,Podoplanin and LYVE-1 in mouse aorta and bone marrow-derived macrophages,VEGFC protein level in bone marrow-derived macrophages,VEGFC fluorescence intensity in aortic sinus plaques,LYVE-1 fluorescence intensity around the aortic sinus and in the myocardium gradually increased with time(P＜0.05).In addition,the mRNA level of LYVE-1 in the aorta and the fluorescence intensity of LYVE-1 around the aortic sinus and in the myocardium were the highest at 28 days(P＜0.05),and gradually decreased(P＜0.05).The expression of the other indicators reached the peak at 21 days.To conclude,rAAV9-SP146-C1-VEGFC could effectively transfect bone marrow-derived macrophages and promote lymphatic hyperplasia in atherosclerotic mice.
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OBJECTIVE Based on fibroblast activation protein(FAP)gene promoter as the response element,to develop a new dual luciferase reporting system for the screening of drugs related to myocardial fibrosis.METHODS The promoter fragment of mouse FAP gene was synthesized in vitro and cloned into plasmid psiCHECK2 to replace HSV-TK promoter,and then a new recombinant plasmid psiCHECK2-FAP was obtained.After the recombinant plasmid psiCHECK2-FAP was digested by restriction endonucliase Hind Ⅲ,the product digested was identified by agar-gel electrophoresis and sequencing.After psiCHECK2-FAP was transient transfected into mouse cardiac fibroblasts(MCFs),and continued cultured for 24 h,and MCFs were treated with Ransforming growth factor β1(TGF-β1,5 μg·L-1)or angiotensinⅡ(Ang Ⅱ,1 μmol·L-1)or palmitic acid(PA,100 μmol·L-1)for 0,12,24,48 h,respectively,the double luciferase reporter gene assay was used to detect luciferase activity;After psiCHECK2-FAP was transient transfected into MCFs,the cells were pretreated with Dapa(1 μmol·L-1)for 1 h,and supplemented with TGF-β1(5 μg·L-1)or AngⅡ(1 μmol·L-1)or PA(100 μmol·L-1),continued treatment for 24 h,the double luciferase reporter gene assay was used to detect luciferase activity,and the expression of collagenⅠand collagen Ⅲ protein was detected with Western blotting.RESULTS The recombinant plasmid psiCHECK2-FAP was digested into two fragments by Hind Ⅲ with the expected strip size,and the sequencing results were completely consistent with the theoretical sequence;Compared with control group,the collagenⅠand collagen Ⅲ protein expression were significantly increased by TGF-β1 or Ang Ⅱ or PA in MCFs(P<0.05,P<0.01).However,compared with TGF-β1 or Ang Ⅱ or PA group,the intervention of Dapa significantly alleviated the promoter activity of FAP gene and the expression of collagenⅠand collagen Ⅲ protein(P<0.05,P<0.01);Compared with control group,luciferase activity was significantly increased by TGF-β1 or Ang Ⅱ or PA(P<0.05,P<0.01),reaching the peak at 24 h.Compared with TGF-β1 or AngⅡ or PA group,the intervention of Dapa significantly decreased luciferase activity(P<0.05,P<0.01).CONCLUSSION Based on the promoter of FAP gene as the response element,a noval dual luciferase reporter gene system was established and showed a good sensitivity to the promyocardial fibrosis factor in MCFs,which can provide strategies for the development of antimyocar-dial fibrosis drugs.
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Abstract Objective To verify the performance of the Net Promoter Score (NPS) as a tool to assess parental satisfaction in pediatric intensive care units (PICUs). Methods The authors conducted an observational cross-sectional multicenter study in the PICUs of 5 hospitals in Brazil. Eligible participants were all parents or legal guardians of PICU-admitted children, aged 18 years or over. The NPS was administered together with the EMpowerment of PArents in THe Intensive Care (EMPATHIC-30), used as the gold standard, and a sociodemographic questionnaire. For analysis, the results were dichotomized into values greater than or equal to the median of the tests. The associations between the 2 tools were evaluated and the distribution of their results was compared. Results The parents or legal guardians of 78 PICU-admitted children were interviewed. Of the respondents, 85% were women and 62% were in a private hospital. The median NPS was 10 (IQR, 10-10), and the median EMPATHIC-30 score was 5.7 (IQR, 5.4-5.9). Compared with the gold standard, the NPS had a sensitivity of 100% at all cutoff points, except at cutoff 10, where the sensitivity was slightly lower (97.5%). As for specificity, NPS performance was poorer, with values ranging from 0% (NPS ≥ 5) to 47.4% (NPS = 10). Conclusions NPS proved to be a sensitive tool to assess parental satisfaction, but with poor ability to identify dissatisfied users in the sample.
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MicroRNA-20a-5p (miR-20a-5p) has been shown to function as a tumor promoter factor in several cancers. However, its role in small cell lung cancer (SCLC) remains unclear. In this study, we have made an attempt to measure the tumor tissue levels of miR-20a-5p in patients with SCLC using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The biological function of miR-20a-5p in SCLC cells was investigated in vitro and in vivo studies, including cell proliferation, migration assays and tumorigenicity in nude mice. Meanwhile?we conducted the luciferase reporter assay to verify the biological relationship between miR-20a-5p and CCNG2. The expression of miR-20a-5p was significantly upregulated in human SCLC compared to that in normal tissues. Kaplan-Meier analysis indicated that patients with high expression of miR-20a-5p are closely related with the shorter survival of SCLC. Further, multivariate analysis showed that miR-20a-5p was an independent prognostic factor. Increasing miR-20a-5p expression promotes the proliferation, migration and invasion of the NCI-H446 cells in vitro and in vivo. Dual-luciferase reporter gene assay demonstrated that miR-20a-5p directly targets CCNG2. These findings suggest that miR-20a-5p levels might be a novel diagnostic and prognostic marker of SCLC. Inhibiting miR-20a-5p could be a promising therapeutic strategy for SCLC.
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Background & objectives: Studies have shown that apart from hereditary breast carcinomas, breast cancer susceptibility gene 1 (BRCA1) mutations conferring to its loss are seen in sporadic breast carcinomas (SBC) as well. The aim of the present study was to assess BRCA1 methylation in females presenting at King George’s Medical University, Lucknow, with SBC by both immunohistochemistry (IHC) and methylation PCR with respect to hormonal profile and various morphological prognostic parameters. The primary objective was to look for the association between BRCA1 protein expression and DNA promoter methylation. Methods: 81 mastectomy specimens from SBC of invasive breast carcinoma (no special type) were included in this study. After a detailed morphological assessment, formalin fixed paraffin embedded tissue from a representative tumour area was selected for BRCA1 IHC by heat-mediated antigen retrieval under high pH and DNA extraction and further bisulphate treatment. BRCA1 was studied for methylation by methylated and unmethylated PCR-specific primers. Results: BRCA1 promoter methylation was present in 42/81 (51.9%) participants, with significant BRCA1 protein loss (72.7%; P=0.002). A significant association between BRCA1 loss and hormonal profile was found (P=0.001); maximum in triple negative breast carcinoma (TNBC) (72%; 18/25). Most of the TNBC also harboured methylation (68%). Although not significant grade II and III tumours, lymph vascular invasion, ductal carcinoma in situ, and nodal metastasis (?3) were seen in a higher percentage in methylated tumours. Mortality in SBC was significantly associated with BRCA1 loss (30.3%; P=0.024). Interpretation & conclusions: Study results highlight the concept of “BRCAness” in SBC as well. Hence, we can confer that identification of BRCA1 loss in SBC can make it a perfect candidate for poly ADP- ribose polymerase inhibitors or cisplatin-based therapy like hereditary ones.
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L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.
Subject(s)
Bacillus licheniformis/metabolism , Asparaginase/genetics , Bacillus/genetics , Protein Sorting Signals , Promoter Regions, Genetic/genetics , Bacillus subtilis/genetics , Bacterial ProteinsABSTRACT
Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1, 4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.
Subject(s)
Peptide Elongation Factors/genetics , Plant Proteins/metabolism , Periploca/metabolism , Rubber , Plant Breeding , Cloning, MolecularABSTRACT
Objective To explore the relationship between scavenger receptor class B member 1 (SCARB1) gene promoter methylation and the pathogenesis of coronary artery disease. Methods A total of 120 patients with coronary heart disease treated in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from December 2018 to May 2020 were selected as the case group,while 140 gender and age matched healthy participants were randomly selected as the control group for a case-control study.The methylation status was detected by high-throughput target sequencing after bisulfite converting,and the methylation of CpG sites in the promoter region of SCARB1 gene was compared between the two groups. Results The case group showed higher methylation level of SCARB1+67 and lower methylation level of SCARB1+134 than the control group (both P<0.001),and the differences remained statistically significant in men (both P<0.001) and women (both P<0.001).The overall methylation level in the case group was lower than that in the control group [(80.27±2.14)% vs.(81.11±1.27)%;P=0.006],while this trend was statistically significant only in men (P=0.002). Conclusion The methylation of SCARB1 gene promotor is associated with the pathogenesis and may participate in the occurrence and development of coronary heart disease.
Subject(s)
Male , Humans , Female , Methylation , Case-Control Studies , China , Coronary Artery Disease/genetics , Promoter Regions, Genetic , DNA Methylation , Scavenger Receptors, Class B/geneticsABSTRACT
The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.
Subject(s)
Panax/genetics , Promoter Regions, Genetic , Agrobacterium tumefaciens/genetics , Computational Biology , Cloning, MolecularABSTRACT
Objective @#To investigate the relevance between vitamin D receptor ( VDR) gene promoter methylation level and idiopathic hypocitraturia of the Bai nationality in Yunnan province．@*Methods @#Fifteen patients with idiopathic hypocitrouria of the Bai nationality in Yunnan province with double dominant expression (FF type) of single nucleotide polymorphism shot (SNP shot) rs2228570 (Fok Ⅰ ) genotype were selected as the experimental group. Fifteen people of the Bai nationality in Yunnan province with normal content of urinary citric acid were the control group．First，blood samples were taken from both groups．Next，the blood samples were treated with sulfites，RNA products of each sample were obtained by PCR amplification and in vitro transcription of T7 DNA polymerase．Then the corresponding RNA fragments were digested by base-specific enzymes．Finally the degree of methylation at each test site was obtained through the EpiTYPER procedure. @*Results@#In the statistical results of DNA methylation level，the methylation level of VDR Ⅰ fragment experimental group was 4. 136% ( 1. 655% ，5. 152% ) which was higher than 1. 261% (0. 827% ，1. 930% ) in the control group，and the difference was statistically significant (P ＜0. 001) ．Among the 33 CpG sites on VDR Ⅰ fragment，there were significant differences in DNA methylation levels of CPG-5 (F = 8. 831，P = 0. 008) and CpG-8 (F = 16. 155，P = 0. 001) between the experimental group and the control group．@*Conclusion@#The increased methylation level of VDR gene promoter is related with idiopathic hypocitric of the Bai nationality of Yunnan province．And compared with the normal Bai nationality people，the DNA methylation level of VDR gene promoter significantly increased in the Bai nationality patients with FF type of VDR gene SNP shot Fok Ⅰ .
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β-lactam antibiotics are a class of antibiotics widely used in clinical treatment. The antibacterial mechanism of them is to inhibit the cell wall synthesis of bacteria. However, the widespread of β-lactam resistance genes among bacteria has posed a great challenge to the use of β-lactam antibiotics. One of the main genes encoding β-lactamase is blaTEM, and 243 subtypes of it have been identified to date. Studying blaTEM gene is of great significance in antibiotic resistance research. This review focused on the discovery, structure, promoter, distribution, spread, antibacterial mechanism and research status of blaTEM gene, hoping to provide reference for further research on the subtypes of β-lactam resistance genes in bacteria.
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Objective:To investigate the distribution of integrons and plasmid-mediated quinolone resistance (PMQR) genes in clinical isolates of Klebsiella aerogenes and to analyze the relationship between integrons and bacterial resistance to antimicrobial agents. Methods:Ninety-one Klebsiella aerogenes strains isolated from clinical samples in the Fengxian District Central Hospital from November 2015 to March 2021 were used in this study. Class 1 and class 2 integron-integrase genes ( intI1 and intI2) and PMQR genes were screened by PCR. The types of promoters and gene cassette arrays of variable regions were determined by sequencing. Besides, the relationship between integrons and antimicrobial resistance was analyzed. Results:The resistance rate of the 91 Klebsiella aerogenes isolates to aztreonam was more than 40.00% and the resistance rates to other commonly used antimicrobial agents were less than 35.00%. Among the 91 isolates, 30 carried the intI1 gene, while none of them carried the intI2 gene. Seven class 1 integron gene cassette arrays of variable regions were detected and the gene cassette array of aac(6′)-11 C- ΔereA2- IS1247- aac3- arr- ΔereA2 was detected in Klebsiella aerogenes. PcH1 with weak activity was the predominant variable region promoter of class 1 integrons. The detection rates of intI1-positive and intI1-negative isolates in ICU, neurosurgery and other clinical departments were statistically different ( P<0.05). The resistance rate of intI1-positive isolates to some commonly used antibiotics was significantly higher than that of intI1-negative isolates ( P<0.05). qnrS gene was the prevalent PMQR gene. The detection rates of integrons and PMQR genes in Klebsiella aerogenes isolates was low except for the strains isolated in 2016. Conclusions:Antimicrobial resistance in Klebsiella aerogenes was closely related to integrons. The distribution of integrons in Klebsiella aerogenes strains isolated from different clinical departments was different, and the monitoring of drug-resistant strains should be strengthened in ICU and neurosurgery. The resistance to quinolones in Klebsiella aerogenes strains in this region was mainly related to qnrS gene.
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Objective To analyze the transcription factor binding sites(TFBS)in the promoter region of E3 ubiquitin protein ligase 1(MIB1)gene in zebrafish,the types of MIB1 interacting genes and proteins and their roles in signaling pathways,and to investigate the regulatory mode and potential function of MIB1 gene.Methods Non-coding RNA(ncRNA)was predicted by National Genomics Data Center(NGDC).Alggen and AnimalTFDB online software were used to predict the TFBS types of MIB1 gene.GeneMANIA and STRING were used to analyze the interacting genes and proteins of MIB1.The related data were obtained through DA-VID website,and gene ontology(GO)visual analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)metabolic pathway analysis were performed.Results ncRNA could be transcribed from the promot-er region and 5'untranslated region of MIB1 gene.A total of 121 TFBS were obtained by prediction.P53 tran-scription factor could bind to the promoter region of MIB1 gene and interact with MIB1 protein.A total of 6 co-expressed genes of MIB1 were predicted online,and 20 interacting genes were screened.GO visual analysis showed that MIB1 and its interaction genes had functions in regulating the growth and differentiation of cells,tissues and organs and regulating the NOTCH signaling pathway in the biological process,and were mainly enriched in the cytoplasmic perinuclear region,cell membrane,postsynaptic dense area and so on.It had molec-ular functions such as binding NOTCH proteins and PDZ domain proteins.KEGG metabolic pathway analysis showed that MIB1 and its interacting genes were involved in 4 metabolic pathways.Conclusion MIB1 con-tains a variety of TFBS,and affects a variety of biological processes such as cell carcinogenesis and immune regulation by interacting with specific transcription factors.MIB1 may also play an important role in cell growth regulation,hematopoietic stem cell differentiation,embryonic development and neuronal information transmission through the mediation of its interacting genes and proteins.
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Tripartite motif containing protein 7 (TRIM7), as a member of the E3 ubiquitin ligase TRIM family, plays an important regulatory role in immune regulation, metabolism and other physiological processes. The aberrant expression of TRIM7 is closely related to the development and progression of hepatocellular carcinoma (HCC) and it shows a complex regulatory role. However, the regulatory mechanism for the expression of TRIM7 in HCC remains unknown. In this study, multiple online databases were used to analyze the expression of TRIM7 in HCC and data indicated that TRIM7 expression was upregulated in HCC and correlated to poor prognosis. Subsequently, the transcription factor binding sites in the TRIM7 promoter region were analyzed using UCSC and JASPAR databases, and the results showed that TRIM7 promoter contains four SP1 binding sites. In this work, we demonstrated that SP1 could directly bind to its binding sites in TRIM7 promoter and positively regulate the transcriptional activity driven by the TRIM7 promoter using dual luciferase reporter experiments and the ChIP-PCR method. Moreover, our results also showed SP1 overexpression upregulated the expression of TRIM7 at both mRNA and protein levels (P<0. 01),and SP1 inhibitor, mithramycin A, could reverse the activated effect of SP1 on TRIM7 expression (P<0. 01). In conclusion, this study preliminarily reveals the regulatory mechanism of TRIM7 upregulation in HCC, which provides an important theoretical basis for further study of the gene function, early diagnosis and targeted therapy.
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OBJECTIVE To investigate the improve-ment functions of flavonoid compounds on temozolomide(TMZ)-,aging-or AD model-induced dysregulation of hip-pocampal NSC lineage progression,retardancy of den-dritic spine maturation in new-born neurons,as well as impairment of hippocampal-related learning and memory.METHODS We applied 30-week-old neural stem cell(NSC)specific promoter Nestin-GFP and NestinCreERT2:Rosa26-LSL-tdTomato transgenic mice and 16-week-old AD model 5XFAD transgenic mice,together with hippo-campal microinjection(ih),endogenous fluorescence trac-ing and immunofluorescent staining.RESULTS Both fla-vonoid compound A and its functional derivative flavo-noid compound B dose-dependently improved TMZ-,aging-or AD-induced defects of hippocampal NSC lin-eage progression and the maturation of dendritic spines of newborn neurons,thereby improving hippocampus related learning and memory.CONCLUSION This paper provides a new idea and treatment strategy for the devel-opment of new flavonoids that can promote neurogene-sis for neurodegenerative diseases and aging.
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Overexpression of Krüppel like factor 2 (Klf2) or Klf7 inhibits adipocyte formation. However, it remains unclear whether Klf2 regulates klf7 expression in adipose tissue. In this study, oil red O staining and Western blotting were employed to study the effect of Klf2 overexpression on the differentiation of chicken preadipocytes. The results showed that Klf2 overexpression inhibited the differentiation of chicken preadipocytes induced by oleate and the expression of pparγ, while promoted klf7 expression in chicken preadipocytes. Spearman correlation analysis was used to study the correlation between the expression data of klf2 and klf7 in the adipose tissue of both human and chicken. The results showed that there was a significantly positive correlation between the expression of klf2 and klf7 in adipose tissues (r > 0.1). Luciferase reporter assay showed that overexpression of Klf2 significantly promoted the activity of chicken klf7 promoter (-241/-91, -521/-91, -1 845/-91, -2 286/-91, -1 215/-91; P < 0.05). In addition, the activity of klf7 promoter (-241/-91) reporter in chicken preadipocytes was significantly positively correlated with the amount of klf2 overexpression plasmid transfected (Tau=0.917 66, P=1.074×10-7). Moreover, Klf2 overexpression significantly promoted the mRNA expression of klf7 in chicken preadipocytes (P < 0.05). In conclusion, upregulation of klf7 expression might be one of the pathways that Klf2 inhibits chicken adipocyte differentiation, and the sequence from -241 bp to -91 bp upstream chicken klf7 translation start site might mediate the regulation of Klf2 on klf7 transcription.
Subject(s)
Animals , Humans , Chickens/genetics , Kruppel-Like Transcription Factors/metabolism , Transcription Factors/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolismABSTRACT
AIM: To analyze the relationship between rs128912 single nucleotide polymorphism(SNP)in the promoter region of Toll-like receptor 3(TLR3)gene and cataract in Chinese Han population.METHODS: A total of 263 patients with cataract admitted to our hospital from June 2019 to June 2021 were selected as study group, and 150 patients with lens dislocation were included in control group. Western blotting was used to detect the expression of TLR3 protein in the anterior capsular tissues of lens in the two groups, and direct sequencing method was applied to analyze the polymorphism of rs128912 locus in the promoter region of TLR3 gene. The expression of peripheral blood TLR3 mRNA of patients with different genotypes was detected by real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS: The expression level of TLR3 protein in the anterior capsular tissues in the study group was higher than that in the control group(P<0.05). The frequencies of genotypes(AA, AT, TT)at rs128912 locus in the TLR3 gene promoter region in the study group and the control group were in accordance with Hardy-Weinberg genetic equilibrium, and there were differences in the frequencies of genotypes(AA, AT, TT)and frequencies of alleles(A, T)at rs128912 locus in the TLR3 gene promoter region between both groups(P<0.05). The relative expression level of peripheral blood TLR3 mRNA in patients with TT genotype in the study group was higher than that in patients with AA or AT genotypes(P<0.05).CONCLUSION: The expression of TLR3 protein in anterior capsular tissues of lens of patients with cataract is significantly up-regulated, and rs128912 locus polymorphism in the TLR3 gene promoter region is related to the susceptibility of cataract in Chinese Han population, and people with TT genotype are more prone to cataract.
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OBJECTIVE@#To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia.@*METHODS@#Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene.@*RESULTS@#The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups.@*CONCLUSION@#After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
Subject(s)
Humans , Transduction, Genetic , Genetic Vectors , Hemophilia A/genetics , Transfection , Blood Coagulation Factors/genetics , Lentivirus/geneticsABSTRACT
Objetivo: analisar os fatores associados a sintomas ansiosos e depressivos de alunos do internato interprofissional da Santa Casa de Belo Horizonte. Analisou-se também a autopercepção de aprendizagem e satisfação. Método: estudo transversal no qual analisou-se os fatores associados a sintomas ansiosos e depressivos estimados pelo Transtorno de Ansiedade Generalizada (GAD-7) e pelo Questionário de Saúde do Paciente (PHQ-9) em alunos de um internato interprofissional de enfrentamento à COVID-19. Ao final do internato, analisou-se a percepção do alcance dos objetivos de aprendizagem e a experiência do aluno utilizando Net Promoter Score (NPS). Resultado: entre os 92 alunos analisados, 22 (23,9%) apresentaram escores elevados para sintomas ansiosos e 26 (28,3%) para sintomas depressivos. A frequência de sintomas ansiosos foi maior entre alunos de farmácia quando comparados aos de medicina ou enfermagem (42,9%, 28,9%, 9,7%, respectivamente, p=0,035). Sintomas ansiosos foram menos frequentes entre alunos que sempre tiveram acesso a equipamento de proteção individual (EPI) quando comparados aos demais (17,7% vs. 36,7%; p=0,046). Alunos que tiveram sintomas de COVID-19, quando comparados aos demais, apresentaram maior frequência de sintomas ansiosos (44,1% vs. 12,1%; p=0,001) e depressivos (41,2% vs. 20,7%; p=0,035). O atendimento a pacientes com COVID-19 não esteve associado a sintomas depressivos nem ansiosos. Observou-se alto nível de percepção do alcance dos objetivos de aprendizagem, maior entre estudantes de enfermagem. O escore geral do NPS foi de 70, com maior frequência de promotores entre alunos de enfermagem (90%), quando comparados aos de farmácia (67%) e medicina (62%). Conclusão: sintomas ansiosos estiveram associados à categoria profissional, acesso a EPI e história prévia de sintomas de COVID-19.A percepção do alcance de objetivos propostos foi elevada e o escore NPS foi satisfatório, com maior proporção de promotores na enfermagem
Objective: to analyze the factors associated with symptoms of anxiety and depression among interprofessional students at Santa Casa de Belo Horizonte. Self-perception of learning and satisfaction were also analyzed. Methods: cross-sectional study that analyzed the factors associated with symptoms of anxiety and depression estimated by the Generalized Anxiety Disorder 7-item (GAD-7) and Patient Health Questionnaire-9 (PHQ-9). At the end of the internship, the perception of achievement of learning objectives and the student's experience were analyzed using the Net Promoter Score (NPS). Results: among the 92 students analyzed, 22 (23,9%) had high scores for anxiety symptoms and 26 (28,3%) for depressive symptoms. The frequency of anxious symptoms was higher among pharmacy students when compared to medicine or nursing students (42.9%, 28.9%, 9.7%, respectively, p=0.035). Anxiety symptoms were less frequent among students who always had access to personal protective equipment (PPE) when compared to the others (17.7% vs. 36.7%; p=0.046). Students who had symptoms of Covid-19, when compared to the others, had a higher frequency of anxiety (44.1% vs. 12.1%; p=0.001) and depressive symptoms (41.2% vs. 20.7%; p =0.035). Caring for Covid-19 patients was not associated with depression or anxiety. There was a high level of perception of achievement of learning objectives, higher among nursing students. Overall NPS score was 70, with a higher frequency of promoters among nursing students (90%), when compared to those in pharmacy (67%) or medicine (62%). Conclusion: anxiety symptoms were associated with the professional category, access to PPE, and previous history of Covid-19. The perception of achievement of learning objectives was high and the NPS score was satisfactory, with more promoters among nursing students