利用斑马鱼模型评价防风多糖的免疫调节作用,并利用转录组测序和实时荧光定量PCR技术探讨其作用机制。以免疫细胞荧光标记的转基因斑马鱼Tg(lyz:DsRed)为实验动物,选用长春瑞滨诱导免疫低下模型,评价防风多糖对斑马鱼体内巨噬细胞密度...利用斑马鱼模型评价防风多糖的免疫调节作用,并利用转录组测序和实时荧光定量PCR技术探讨其作用机制。以免疫细胞荧光标记的转基因斑马鱼Tg(lyz:DsRed)为实验动物,选用长春瑞滨诱导免疫低下模型,评价防风多糖对斑马鱼体内巨噬细胞密度和分布的影响。以野生型AB系斑马鱼为实验动物,采用中性红和苏丹黑B染色法,检测防风多糖对斑马鱼体内巨噬细胞和中性粒细胞数目的影响;采用DAF-FM DA荧光探针法,检测各组斑马鱼体内NO含量的变化;ELISA法检测斑马鱼体内IL-1β和IL-6的含量变化。利用转录组测序分析空白对照组、模型组和防风多糖处理组斑马鱼的差异表达基因,通过Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析其免疫调节作用机制,并利用RT-qPCR法验证关键基因的表达水平。结果显示,防风多糖能显著增加斑马鱼免疫细胞密度,增加巨噬细胞和中性粒细胞数目,并降低免疫低下斑马鱼体内NO、IL-1β和IL-6含量。转录组测序分析结果显示防风多糖通过影响Toll样受体通路和单纯疱疹感染通路上免疫相关基因的表达水平,进而影响下游的细胞因子以及干扰素的释放,完成T细胞的活化过程,发挥调节机体免疫活性的作用。展开更多
The highbush blueberry（Vaccinium corymbosum）,Duke,was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.Mega 4,CLC Sequence Viewer 6 software,and quantitative PCR we...The highbush blueberry（Vaccinium corymbosum）,Duke,was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.Mega 4,CLC Sequence Viewer 6 software,and quantitative PCR were employed for bioinformatics and expression analyses of the basic helix-loop-helix（BHLH）transcription factors of the sequencing library.The results showed that 28.38 gigabytes of valid data were obtained from transcriptome sequencing and were assembled into 108 033 unigenes.Functional annotation showed that 32 244 unigenes were annotated into Clusters of Orthologous Groups（COG）and Gene Ontology（GO）databases,whereas the rest of the 75 789 unigenes had no matching information.By using COG and GO classification tools,sequences with annotation information were divided into 25 and 52 categories,respectively,which involved transport and metabolism,transcriptional regulation,and signal transduction.Analysis of the transcriptome library identified a total of 59 BHLH genes.Sequence analysis revealed that 55 genes of that contained a complete BHLH domain.Furthermore,phylogenetic analysis showed that BHLH genes of blueberry（Duke）could be divided into 13 sub-groups.PCR results showed that 45 genes were expressed at various developmental stages of buds,stems,leaves,flowers,and fruits,suggesting that the function of BHLH was associated with the development of different tissues and organs of blueberry,Duke.The present study would provided a foundation for further investigations on the classification and functions of the blueberry BHLH family.展开更多
Background:Tumor heterogeneity is contributed by tumor cells and the microenvironment.Dynamics of tumor heterogeneity during colorectal cancer(CRC)progression have not been elucidated.Methods:Eight single-cell RNA seq...Background:Tumor heterogeneity is contributed by tumor cells and the microenvironment.Dynamics of tumor heterogeneity during colorectal cancer(CRC)progression have not been elucidated.Methods:Eight single-cell RNA sequencing(scRNA-seq)data sets of CRC were included.Milo was utilized to reveal the differential abundance of cell clusters during progression.The differentiation trajectory was imputed by using the Palantir algorithm and metabolic states were assessed by using scMetabolism.Three spatial transcription sequencing(ST-seq)data sets of CRC were used to validate cell-type abundances and colocalization.Cancer-associated regulatory hubs were defined as communication networks affecting tumor biological behaviors.Finally,quantitative reverse transcription polymerase chain reaction and immunohistochemistry staining were performed for validation.Results:TM4SF1t,SOX4t,and MKI67t tumor cells;CXCL12t cancer-associated fibroblasts;CD4t resident memory T cells;Treg;IgAt plasma cells;and several myeloid subsets were enriched in stage IV CRC,most of which were associated with overall survival of patients.Trajectory analysis indicated that tumor cells from patients with advanced-stage CRC were less differentiated,when metabolic heterogeneity showed a highest metabolic signature in terminal states of stromal cells,T cells,and myeloid cells.Moreover,ST-seq validated cell-type abundance in a spatial context and also revealed the correlation of immune infiltration between tertiary lymphoid structures and tumors followed by validation in our cohort.Importantly,analysis of cancer-associated regulatory hubs revealed a cascade of activated pathways including leukocyte apoptotic process,MAPK pathway,myeloid leukocyte differentiation,and angiogenesis during CRC progression.Conclusions:Tumor heterogeneity was dynamic during progression,with the enrichment of immunosuppressive Treg,myeloid cells,and fibrotic cells.The differential state of tumor cells was associated with cancer staging.Assessment of cancer-associated regulatory hubs 展开更多
A number of internal signals are required for seed germination.However,the precise signalling responses in the initial imbibition of seed germination are not yet fully understood in rice.In this study,the RNA sequenci...A number of internal signals are required for seed germination.However,the precise signalling responses in the initial imbibition of seed germination are not yet fully understood in rice.In this study,the RNA sequencing(RNA-Seq)approach was conducted in 8 h imbibed seeds to understand the signalling responses in the initial imbibition of rice seed germination.A total of 563 differentially expressed genes(DEGs)with at least 4-fold change were identified in 8 h imbibed seeds compared to dry seeds.MapMan analysis revealed that the majority of signalling response-related DEGs were hormone-and transcription factor-related genes,in which the largest number of DEGs belong to the AP2-domain-containing regulators,and their expressions were significantly induced in the initial imbibition of seed germination in rice.Moreover,at least five AP2-domain-containing transcription factor OsDREBs were identified in the initial imbibition of rice seed germination,and the expressions of 251 DEGs were putatively regulated by OsDREBs through the dehydration-responsive element(DRE)cis-element assay.It suggested that the OsDREBs might play important roles in the regulation of initial seed imbibition in rice.The identified genes provide a valuable resource to study the signalling regulation of seed germination in the future.展开更多
The xenobiotic transcription factor cap‘n’collar isoform C(CncC)is considered the central regulator of antioxidant and detoxification genes.Previous research indicated that CncC regulates three-phase enzymes respons...The xenobiotic transcription factor cap‘n’collar isoform C(CncC)is considered the central regulator of antioxidant and detoxification genes.Previous research indicated that CncC regulates three-phase enzymes responsible for insecticide resistance.In this study,the SlituCncC gene from Spodoptera litura was obtained and characterized.Quantitative polymerase chain reaction(qPCR)analysis showed that SlituCncC was expressed in all developmental stages and tissues,but was highly expressed in 3rd-and 4th-instar larvae,and in the Malpighian tubule,fat body,and midgut.In addition,SlituCncC was up-regulated and more highly induced with indoxacarb treatment in the indoxacarb-resistant strains compared with the susceptible strain.RNA interference-mediated gene silencing of SlituCncC significantly increased mortality of S.liura when exposed to in-doxacarb.Furthermore,comparative transcriptome analysis showed that 842 genes were down-regulated and 127 genes were up-regulated in SlituC"ncC knockdown S.litura.Further analysis indicated that 18 three-phase enzymes were identified in the down-regulated genes,of which seven were associated with indoxacarb resistance in S.litura.qPCR analysis confirmed that expression of six of these seven genes was consistent with RNA se-quencing data.All six detoxification genes were induced by indoxacarb,and the expression patterns were similar to that of SlituCncC.Finally,the CneC-Maf binding site was predicted in all six gene promoters.This study indicates that the transcription factor SlituCncC may regulate multiple detoxification genes that mediate indoxacarb resistance in S.litura.展开更多
Ciliary neurotrophic factor (CNTF) is the first neurokine to be found out for the potency to promote motoneuron survival in vitro and in vivo, and it does play a role in the development and regeneration of the nervous...Ciliary neurotrophic factor (CNTF) is the first neurokine to be found out for the potency to promote motoneuron survival in vitro and in vivo, and it does play a role in the development and regeneration of the nervous system. CNTF has been found up mainly in the astrocyte and Schwann cell. Its gene is located in the proximal part of long arm of chromosome 11, and apparently heterogenous to the gene of neurotrophins, there is展开更多
Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were id...Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were identified and captured by interphase fluorescence in situ hybridisation(iFISH).Single cell whole exome sequencing(WES)was performed and the corresponding bioinformatics data were analysed.OCT4+cells were successfully detected in peripheral blood collected from all four Stage I lung cancer patients.Moreover,the tumour mutational burden(TMB)values observed for OCT4+samples from the same patients were slightly smaller than those of the OCT4−samples;the difference was not statistically significant(P>0.05).Thirteen and six characteristic mutations were found in negative samples and positive samples,respectively.The findings indicate that this methodology provides a potential diagnostic index for the early detection of NSCLC.展开更多
The accurate annotation of transcription start sites(TSSs)and their usage are critical for the mechanistic understanding of gene regulation in different biological contexts.To fulfill this,specific high-throughput exp...The accurate annotation of transcription start sites(TSSs)and their usage are critical for the mechanistic understanding of gene regulation in different biological contexts.To fulfill this,specific high-throughput experimental technologies have been developed to capture TSSs in a genome-wide manner,and various computational tools have also been developed for in silico prediction of TSSs solely based on genomic sequences.Most of these computational tools cast the problem as a binary classification task on a balanced dataset,thus resulting in drastic false positive predictions when applied on the genome scale.Here,we present Dee Re CT-TSS,a deep learningbased method that is capable of identifying TSSs across the whole genome based on both DNA sequence and conventional RNA sequencing data.We show that by effectively incorporating these two sources of information,Dee Re CT-TSS significantly outperforms other solely sequence-based methods on the precise annotation of TSSs used in different cell types.Furthermore,we develop a meta-learning-based extension for simultaneous TSS annotations on 10 cell types,which enables the identification of cell type-specific TSSs.Finally,we demonstrate the high precision of DeeReCT-TSS on two independent datasets by correlating our predicted TSSs with experimentally defined TSS chromatin states.The source code for Dee Re CT-TSS is available at https://github.-com/Joshua Chou2018/Dee Re CT-TSS_release and https://ngdc.cncb.ac.cn/biocode/tools/BT007316.展开更多
文摘利用斑马鱼模型评价防风多糖的免疫调节作用,并利用转录组测序和实时荧光定量PCR技术探讨其作用机制。以免疫细胞荧光标记的转基因斑马鱼Tg(lyz:DsRed)为实验动物,选用长春瑞滨诱导免疫低下模型,评价防风多糖对斑马鱼体内巨噬细胞密度和分布的影响。以野生型AB系斑马鱼为实验动物,采用中性红和苏丹黑B染色法,检测防风多糖对斑马鱼体内巨噬细胞和中性粒细胞数目的影响;采用DAF-FM DA荧光探针法,检测各组斑马鱼体内NO含量的变化;ELISA法检测斑马鱼体内IL-1β和IL-6的含量变化。利用转录组测序分析空白对照组、模型组和防风多糖处理组斑马鱼的差异表达基因,通过Gene Ontology(GO)和Kyoto Encyclopedia of Genes and Genomes(KEGG)富集分析其免疫调节作用机制,并利用RT-qPCR法验证关键基因的表达水平。结果显示,防风多糖能显著增加斑马鱼免疫细胞密度,增加巨噬细胞和中性粒细胞数目,并降低免疫低下斑马鱼体内NO、IL-1β和IL-6含量。转录组测序分析结果显示防风多糖通过影响Toll样受体通路和单纯疱疹感染通路上免疫相关基因的表达水平,进而影响下游的细胞因子以及干扰素的释放,完成T细胞的活化过程,发挥调节机体免疫活性的作用。
基金supported by the National Natural Science Foundation of China (31301754)the Chinese Academy of Agricultural Sciences-Agricultural Science and Technology Innovation Program (CAAS-ASTIP)the Cultivation Plan for Youth Agricultural Science and Technology Innovative Talents of Liaoning Province, China (2015059)
文摘The highbush blueberry（Vaccinium corymbosum）,Duke,was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.Mega 4,CLC Sequence Viewer 6 software,and quantitative PCR were employed for bioinformatics and expression analyses of the basic helix-loop-helix（BHLH）transcription factors of the sequencing library.The results showed that 28.38 gigabytes of valid data were obtained from transcriptome sequencing and were assembled into 108 033 unigenes.Functional annotation showed that 32 244 unigenes were annotated into Clusters of Orthologous Groups（COG）and Gene Ontology（GO）databases,whereas the rest of the 75 789 unigenes had no matching information.By using COG and GO classification tools,sequences with annotation information were divided into 25 and 52 categories,respectively,which involved transport and metabolism,transcriptional regulation,and signal transduction.Analysis of the transcriptome library identified a total of 59 BHLH genes.Sequence analysis revealed that 55 genes of that contained a complete BHLH domain.Furthermore,phylogenetic analysis showed that BHLH genes of blueberry（Duke）could be divided into 13 sub-groups.PCR results showed that 45 genes were expressed at various developmental stages of buds,stems,leaves,flowers,and fruits,suggesting that the function of BHLH was associated with the development of different tissues and organs of blueberry,Duke.The present study would provided a foundation for further investigations on the classification and functions of the blueberry BHLH family.
基金supported by the National Key Research and Development Program of China[grant number 2022YFA1304000]the National Natural Science Foundation of China Key Joint Project[grant number U21A20344]+5 种基金the National Natural Science Foundation of China[grant number 81970452]the Program of Guangdong Provincial Clinical Research Center for Digestive Diseases[grant number 2020B1111170004]the Science and Technology Program of Shenzhen,China[grant number JCYJ20190807161807867]the Starting Funding of Faculty from Sun Yat-sen University[grant number 2021276]the Regional Joint Project for Basic and Applied Basic Research Fund of Guangdong Province[grant number 2022A1515111043]the Science and Technology Planning Project of Guangzhou City[grant number 2023A04J01601],and National Key Clinical Discipline.
文摘Background:Tumor heterogeneity is contributed by tumor cells and the microenvironment.Dynamics of tumor heterogeneity during colorectal cancer(CRC)progression have not been elucidated.Methods:Eight single-cell RNA sequencing(scRNA-seq)data sets of CRC were included.Milo was utilized to reveal the differential abundance of cell clusters during progression.The differentiation trajectory was imputed by using the Palantir algorithm and metabolic states were assessed by using scMetabolism.Three spatial transcription sequencing(ST-seq)data sets of CRC were used to validate cell-type abundances and colocalization.Cancer-associated regulatory hubs were defined as communication networks affecting tumor biological behaviors.Finally,quantitative reverse transcription polymerase chain reaction and immunohistochemistry staining were performed for validation.Results:TM4SF1t,SOX4t,and MKI67t tumor cells;CXCL12t cancer-associated fibroblasts;CD4t resident memory T cells;Treg;IgAt plasma cells;and several myeloid subsets were enriched in stage IV CRC,most of which were associated with overall survival of patients.Trajectory analysis indicated that tumor cells from patients with advanced-stage CRC were less differentiated,when metabolic heterogeneity showed a highest metabolic signature in terminal states of stromal cells,T cells,and myeloid cells.Moreover,ST-seq validated cell-type abundance in a spatial context and also revealed the correlation of immune infiltration between tertiary lymphoid structures and tumors followed by validation in our cohort.Importantly,analysis of cancer-associated regulatory hubs revealed a cascade of activated pathways including leukocyte apoptotic process,MAPK pathway,myeloid leukocyte differentiation,and angiogenesis during CRC progression.Conclusions:Tumor heterogeneity was dynamic during progression,with the enrichment of immunosuppressive Treg,myeloid cells,and fibrotic cells.The differential state of tumor cells was associated with cancer staging.Assessment of cancer-associated regulatory hubs
基金supported by the National Key Research and Development Plan (Grant No. 2018YFD0100901)the Guangdong Province Key Research and Development Program (Grant No. 2018B020202012)+1 种基金the Guangdong Province Key Laboratory of Plant Molecular Breeding (Grant No. GPKLPMB201903)the Major Scientific Research Projects of General Colleges and Universities of Guangdong Province (Grant No. 2017KTSCX024)
文摘A number of internal signals are required for seed germination.However,the precise signalling responses in the initial imbibition of seed germination are not yet fully understood in rice.In this study,the RNA sequencing(RNA-Seq)approach was conducted in 8 h imbibed seeds to understand the signalling responses in the initial imbibition of rice seed germination.A total of 563 differentially expressed genes(DEGs)with at least 4-fold change were identified in 8 h imbibed seeds compared to dry seeds.MapMan analysis revealed that the majority of signalling response-related DEGs were hormone-and transcription factor-related genes,in which the largest number of DEGs belong to the AP2-domain-containing regulators,and their expressions were significantly induced in the initial imbibition of seed germination in rice.Moreover,at least five AP2-domain-containing transcription factor OsDREBs were identified in the initial imbibition of rice seed germination,and the expressions of 251 DEGs were putatively regulated by OsDREBs through the dehydration-responsive element(DRE)cis-element assay.It suggested that the OsDREBs might play important roles in the regulation of initial seed imbibition in rice.The identified genes provide a valuable resource to study the signalling regulation of seed germination in the future.
基金the National Natural Science Foundation of China(31801764)the Natural Science Foundation of Hunan Province,China(2019JJ50230)+2 种基金the Scientific Research Fund of Hunan Provincial Education Department(18B 105)China Postdoctoral Science Foundation(2019M652770)the Youth Fund Project of Hunan Agricultural University(17QN34)。
文摘The xenobiotic transcription factor cap‘n’collar isoform C(CncC)is considered the central regulator of antioxidant and detoxification genes.Previous research indicated that CncC regulates three-phase enzymes responsible for insecticide resistance.In this study,the SlituCncC gene from Spodoptera litura was obtained and characterized.Quantitative polymerase chain reaction(qPCR)analysis showed that SlituCncC was expressed in all developmental stages and tissues,but was highly expressed in 3rd-and 4th-instar larvae,and in the Malpighian tubule,fat body,and midgut.In addition,SlituCncC was up-regulated and more highly induced with indoxacarb treatment in the indoxacarb-resistant strains compared with the susceptible strain.RNA interference-mediated gene silencing of SlituCncC significantly increased mortality of S.liura when exposed to in-doxacarb.Furthermore,comparative transcriptome analysis showed that 842 genes were down-regulated and 127 genes were up-regulated in SlituC"ncC knockdown S.litura.Further analysis indicated that 18 three-phase enzymes were identified in the down-regulated genes,of which seven were associated with indoxacarb resistance in S.litura.qPCR analysis confirmed that expression of six of these seven genes was consistent with RNA se-quencing data.All six detoxification genes were induced by indoxacarb,and the expression patterns were similar to that of SlituCncC.Finally,the CneC-Maf binding site was predicted in all six gene promoters.This study indicates that the transcription factor SlituCncC may regulate multiple detoxification genes that mediate indoxacarb resistance in S.litura.
文摘Ciliary neurotrophic factor (CNTF) is the first neurokine to be found out for the potency to promote motoneuron survival in vitro and in vivo, and it does play a role in the development and regeneration of the nervous system. CNTF has been found up mainly in the astrocyte and Schwann cell. Its gene is located in the proximal part of long arm of chromosome 11, and apparently heterogenous to the gene of neurotrophins, there is
基金the National Natural Science Foundation of China(No.81773273)。
文摘Circulating tumour cells(CTCs)were enriched in the peripheral blood of four patients with Stage I non-small cell lung cancer(NSCLC).Octamer-binding transcription factor-4 positive(OCT4+)and negative(OCT4−)CTCs were identified and captured by interphase fluorescence in situ hybridisation(iFISH).Single cell whole exome sequencing(WES)was performed and the corresponding bioinformatics data were analysed.OCT4+cells were successfully detected in peripheral blood collected from all four Stage I lung cancer patients.Moreover,the tumour mutational burden(TMB)values observed for OCT4+samples from the same patients were slightly smaller than those of the OCT4−samples;the difference was not statistically significant(P>0.05).Thirteen and six characteristic mutations were found in negative samples and positive samples,respectively.The findings indicate that this methodology provides a potential diagnostic index for the early detection of NSCLC.
基金supported in part by grants from Office of Research Administration(ORA)at King Abdullah University of Science and Technology(KAUST)(Grant Nos.BAS/1/1624-01-01,FCC/1/197604-01,URF/1/4098-01-01,REI/1/0018-01-01,REI/1/4216-0101,REI/1/4437-01-01,REI/1/4473-01-01,URF/1/4352-01-01,REI/1/4742-01-01,and URF/1/4663-01-01)supported in part by the National Natural Science Foundation of China(Grant No.31970601)+1 种基金the Shenzhen Science and Technology Program(Grant No.KQTD20180411143432337)the Shenzhen Key Laboratory of Gene Regulation and Systems Biology(Grant No.ZDSYS20200811144002008),China。
文摘The accurate annotation of transcription start sites(TSSs)and their usage are critical for the mechanistic understanding of gene regulation in different biological contexts.To fulfill this,specific high-throughput experimental technologies have been developed to capture TSSs in a genome-wide manner,and various computational tools have also been developed for in silico prediction of TSSs solely based on genomic sequences.Most of these computational tools cast the problem as a binary classification task on a balanced dataset,thus resulting in drastic false positive predictions when applied on the genome scale.Here,we present Dee Re CT-TSS,a deep learningbased method that is capable of identifying TSSs across the whole genome based on both DNA sequence and conventional RNA sequencing data.We show that by effectively incorporating these two sources of information,Dee Re CT-TSS significantly outperforms other solely sequence-based methods on the precise annotation of TSSs used in different cell types.Furthermore,we develop a meta-learning-based extension for simultaneous TSS annotations on 10 cell types,which enables the identification of cell type-specific TSSs.Finally,we demonstrate the high precision of DeeReCT-TSS on two independent datasets by correlating our predicted TSSs with experimentally defined TSS chromatin states.The source code for Dee Re CT-TSS is available at https://github.-com/Joshua Chou2018/Dee Re CT-TSS_release and https://ngdc.cncb.ac.cn/biocode/tools/BT007316.
