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Objective To investigate the expression profile of long non - coding RNAs(lncRNAs) in acute kidney injury (AKI) rats upon astragaloside IV(AS - IV) treatment. Methods Eight male SD rats were selected and randomly divided into two groups. AKI model group(group 1):4 rats were subjected to ischemia - reperfusion(I/ R);AS - Ⅳ pretreatment group (group 2):4 rats were orally administered AS - Ⅳ for 7 days prior to I/ R. Renal tissues were collected after I/ R treatment of 24h,total RNA was extracted from renal tissues and tested for quality. Sequencing experiments were carried out after being qualified. The threshold set for up - and down - regulated genes was a fold change(group1 / group2)≥2 and P≤0. 05. Afterwards,GO analysis and KEGG analysis were used to determine the roles that these differentially expressed mRNAs played in these GO terms or pathways. Results Two hundred and thirty - two lncRNAs were differentially expressed(127 lncRNAs were up - regulated and 105 lncRNAs were down -regulated). Three hundred and forty - one mRNAs were differentially expressed(178 mRNAs were up - regulated and 163 mRNAs were down - regulated). GO analysis indicated that differentially expressed mRNAs were mainly involved in biological processes such as nucleosome assembly,chromatin assembly,nucleosome organization,chromatin assembly or disassembly and DNA conformation change. GO analysis indicated that differentially expressed mRNAs were mainly involved in cellular components such as nucleosome,protein - DNA complex,nuclear chromatin,chromatin and nuclear nucleosome. GO analysis indicated that differentially expressed mRNAs were mainly involved in molecular functions such as protein heterodimerization activity,protein dimerization activity,DNA binding,nucleic acid binding. Signal pathway analysis indicated that lncRNAs and mRNAs were involved in systemic lupus erythematosus,alcoholism and viral carcinogenesis. Conclusion LncRNAs expression differed significantly in renal tissues of AKI model group and AS - Ⅳ preconditioning group. Study of the biological functions and pathways of these lncRNAs indicated that they may be involved in the mechanism of AS - Ⅳ ameliorated ischemic acute renal injury.
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Enterovirus 71 (EV71) is one of the important pathogens of hand, foot and mouth disease (HFMD) in infants and young children. It is also a very important and common virus after poliovirus which is associated with severe acute neurological disease. Mutations or spatial structure changes in untranslated regions (UTRs) may affect the ability of the virus to translate and replicate, as well as the affinity of the virus for cellular tissue, and even lead to changes in virulence. At present, the pathogenic mechanism of EV71 is still unknown, and the study of untranslated regions is an indispensable part. Here we briefly review the development of the study of basic structure and function of EV71 untranslated regions in recent years.
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OBJECTIVE@#To study evolutionary relationship of the 5'untranslated regions (5'UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution (1977-2000) comparing to the DEN3 prototype (H87).@*METHODS@#The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced. Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing to the H87 prototype were done in human (HepG2) and the mosquito (C6/36) cell lines.@*RESULTS@#Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90T, and two minor SNPs including A109G and A112G were found. The C90T of Thai DEN3, Bangkok isolates was shown predominantly before 1977. Five Thai DEN3 candidates with the predominant C90T were shown to replicate in human (HepG2) and the mosquito (C6/36) cell lines better than the H87 prototype. However, their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human.@*CONCLUSIONS@#Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high-order secondary structure and the predominant C90T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.
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Objective:To study evolutionary relationship of the 5’untranslated regions (5’UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution (1977-2000) comparing to the DEN3 prototype (H87).Methods:The 5’UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced. Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing to the H87 prototype were done in human (HepG2) and the mosquito (C6/36) cell lines.Results:Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high-order secondary structure of 5’UTRs and three SNPs including the predominant C90T, and two minor SNPs including A109G and A112G were found. The C90T of Thai DEN3, Bangkok isolates was shown predominantly before 1977. Five Thai DEN3 candidates with the predominant C90T were shown to replicate in human (HepG2) and the mosquito (C6/36) cell lines better than the H87 prototype. However, their highly conserved sequences as well as SNPs of the 5’UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high-order secondary structure and the predominant C90T of the 5’UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.
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Objective: To study evolutionary relationship of the 5'untranslated regions (5'UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution (1977-2000) comparing to the DEN3 prototype (H87). Methods: The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced. Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing to the H87 prototype were done in human (HepG2) and the mosquito (C6/36) cell lines. Results: Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90T, and two minor SNPs including A109G and A112G were found. The C90T of Thai DEN3, Bangkok isolates was shown predominantly before 1977. Five Thai DEN3 candidates with the predominant C90T were shown to replicate in human (HepG2) and the mosquito (C6/36) cell lines better than the H87 prototype. However, their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human. Conclusions: Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high-order secondary structure and the predominant C90T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.
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Objective: To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris. Methods: The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was transformed into E. coli DH5α to construct a modified eukaryotic vector pPIC9-EDIT. After PCR and sequencing, pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast, the product was then transformed into E. coli DH5α to construct the recombinant expression vector pPIC9-EDIT-LL-37, the latter was transformed into P. pastoris GS115 by spheroplasting and the insert was confirmed by PCR. The bacteriolytic activity to E. coli. DH5α was analyzed to screen the highest expressing strain and to determine the best inducing time and concentration of methanol. The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting. The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-EDIT-LL-37 were compared, and the changes of LL-37 protein expression were determined before and after modification. Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed. Expression of LL-37 gene was confirmed by PCR in P. pastoris after pPIC9-EDIT-LL-37 transformation. The highest expressing strain was identified; the best inducing time was 72 h and the best concentration of methanol was 0.5%. Tricine-SDS-PAGE and Western blotting analysis showed that the expression product was LL-37. The expression level of LL-37 protein increased by 35 times after modification. Conclusion: Modification of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P. pastoris; it is worth to be used in the research of other heterogenous protein.
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Objective To screen and analyze nucleotide variants in 5'-untranslated region(5'-UTR)in voltage-gated sodium channel α1-subunit gene(SCN1A)in patients with Dravet syndrome and to evaluate the association of the variants with disease.Methods Peripheral blood of 24 patients with Dravet syndrome and 100 unrelated normal persons were collected and genomic DNA was extracted.PCR-sequencing of SCN1 A 5'-UTR in these DNA was performed.To evaluate the possibility of mutation inducing disease,bioinformatics analysis was applied to analyze the conservation of the sequences around the mutation site and predict the potential transcription elements.Results The nucleotide variant of 166.642.520G→A in exon 2 was identified in two patients,but not in normal controls.The mutation was a de novo mutation in a patient with early-onset.In the second proband,the mutation was also carried by his clinically asymptomatic mother.The nucleotide site 166.642.520 was moderately conserved in mammals(62.5%).The average nucleotide identity rate between human and other mammals species in the region adjacent to 166.642.520 was 88.5%.Two potential transcription regulatory elements were predicted on the sequence with the mutation of 166.642.520G>A,and only one on the sequence with wild-type.Conclusions The mutation 166.642.520G>A may be associated with Dravet syndrome and further studied should be performed to verify it and demonstrate its pathogenic mechanisms.
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Objective:To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris.Methods:The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was trans- formed into E.coli DH5?to construct a modified eukaryotic vector pPIC9-EDIT.After PCR and sequencing,pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast,the product was then transformed into E.coli DH5?to con- struct the recombinant expression vector pPIC9-EDIT-LL-37,the latter was transformed into P.pastoris GS115 by spheroplas- ting and the insert was confirmed by PCR.The bacteriolytic activity to E.coli.DH5?was analyzed to screen the highest ex- pressing strain and to determine the best inducing time and concentration of methanol.The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting.The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-ED- IT-LL-37 were compared,and the changes of LL-37 protein expression were determined before and after modification.Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed.Expression of LL-37 gene was confirmed by PCR in P.pastoris after pPIC9-EDIT-LL-37 transformation.The highest expressing strain was identified;the best inducing time was 72 h and the best concentration of methanol was 0.5%.Tricine-SDS-PAGE and Western blotting analysis showed that the ex- pression product was LL-37.The expression level of LL-37 protein increased by 35 times after modification.Conclusion:Modifi- cation of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P.pastoris;it is worth to be used in the research of other heterogenous protein.