Following spinal cord ischemia/reperfusion injury,an endogenous damage system is immediately activated and participates in a cascade reaction.It is difficult to interpret dynamic changes in these pathways,but the exam...Following spinal cord ischemia/reperfusion injury,an endogenous damage system is immediately activated and participates in a cascade reaction.It is difficult to interpret dynamic changes in these pathways,but the examination of the transcriptome may provide some information.The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome.We used DNA microarrays to measure the expression levels of dynamic evolution-related m RNA after spinal cord ischemia/reperfusion injury in rats.The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours.The simple ischemia group and sham group served as controls.After rats had regained consciousness,hindlimbs showed varying degrees of functional impairment,and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups.Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group,and mitigated in the 48-hour reperfusion group.There were 8,242 differentially expressed m RNAs obtained by Multi-Class Dif in the simple ischemia group,24-hour and 48-hour reperfusion groups.Sixteen m RNA dynamic expression patterns were obtained by Serial Test Cluster.Of them,five patterns were significant.In the No.28 pattern,all differential genes were detected in the 24-hour reperfusion group,and their expressions showed a trend in up-regulation.No.11 pattern showed a decreasing trend in m RNA whereas No.40 pattern showed an increasing trend in m RNA from ischemia to 48 hours of reperfusion,and peaked at 48 hours.In the No.25 and No.27 patterns,differential expression appeared only in the 24-hour and 48-hour reperfusion groups.Among the five m RNA dynamic expression patterns,No.11 and No.40 patterns could distinguish normal spinal cord from pathological tissue.No.25 and No.27 patterns could distinguish simple ischemia from ischemia/reperfusion.No.28 pattern could analyze the need for inducing reperfusion injury.The study of specific pathways and functions for different dynamic patterns can provide a theoretical basis for clinical differential diagnosis and treatment of spinal cord ischemia/reperfusion injury.展开更多
The pen shell(Atrina pectinata) is a large wedge-shaped bivalve, which belongs to family Pinnidae. Due to its large and nutritious adductor muscle, it is the popular seafood with high commercial value in Asia-Pacific ...The pen shell(Atrina pectinata) is a large wedge-shaped bivalve, which belongs to family Pinnidae. Due to its large and nutritious adductor muscle, it is the popular seafood with high commercial value in Asia-Pacific countries. However, limiting genomic and transcriptomic data have hampered its genetic investigations. In this study, the transcriptome of A. pectinata was deeply sequenced using Illumina pair-end sequencing technology. After assembling, a total of 127263 unigenes were obtained. Functional annotation indicated that the highest percentage of unigenes(18.60%) was annotated on GO database, followed by 18.44% on PFAM database and 17.04% on NR database. There were 270 biological pathways matched with those in KEGG database. Furthermore, a total of 23452 potential simple sequence repeats(SSRs) were identified, of them the most abundant type was mono-nucleotide repeats(12902, 55.01%), which was followed by di-nucleotide(8132, 34.68%), tri-nucleotide(2010, 8.57%), tetra-nucleotide(401, 1.71%), and penta-nucleotide(7, 0.03%) repeats. Sixty SSRs were selected for validating and developing genic SSR markers, of them 23 showed polymorphism in a cultured population with the average observed and expected heterozygosities of 0.412 and 0.579, respectively. In this study, we established the first comprehensive transcript dataset of A. pectinata genes. Our results demonstrated that RNA-Seq is a fast and cost-effective method for genic SSR development in non-model species.展开更多
High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many ant...High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA(long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix(Xist antisense RNA), chicken LXN(latexin) and GFM1(Gelongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.展开更多
Industrial manufacturing of bioproducts,especially bioethanol,can benefit from high-temperature fermentation,which requires the use of thermotolerant yeast strains.Mitochondrial activity in yeast is closely related to...Industrial manufacturing of bioproducts,especially bioethanol,can benefit from high-temperature fermentation,which requires the use of thermotolerant yeast strains.Mitochondrial activity in yeast is closely related to its over-all metabolism.However,the mitochondrial respiratory changes in response to adaptive thermotolerance are still poorly understood and have been rarely utilized for developing thermotolerant yeast cell factories.Here,adap-tive evolution and transcriptional sequencing,as well as whole-genome-level gene knockout,were used to obtain a thermotolerant strain of Saccharomyces cerevisiae.Furthermore,thermotolerance and bioethanol production efficiency of the engineered strain were examined.Physiological evaluation showed the boosted fermentation ca-pacity and suppressed mitochondrial respiratory activity in the thermotolerant strain.The improved fermentation produced an increased supply of adenosine triphosphate required for more active energy-consuming pathways.Transcriptome analysis revealed significant changes in the expression of the genes involved in the mitochondrial respiratory chain.Evaluation of mitochondria-associated gene knockout confirmed that ADK1,DOC1,or MET7 were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain.Intriguingly,overexpression of DOC1 with TEF1 promoter regulation led to a 10.1%increase in ethanol production at 42℃.The relationships between thermotolerance,mitochondrial activity,and respiration were explored,and a ther-motolerant yeast strain was developed by altering the expression of mitochondrial respiration-related genes.This study provides a better understanding on the physiological mechanism of adaptive evolution of thermotolerance in yeast.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 suggested impaired antitumor immunity and increased metastatic ability during CRC progression.展开更多
基金supported by the National Natural Science Foundation of China,No.81350013 and 31572217
文摘Following spinal cord ischemia/reperfusion injury,an endogenous damage system is immediately activated and participates in a cascade reaction.It is difficult to interpret dynamic changes in these pathways,but the examination of the transcriptome may provide some information.The transcriptome reflects highly dynamic genomic and genetic information and can be seen as a precursor for the proteome.We used DNA microarrays to measure the expression levels of dynamic evolution-related m RNA after spinal cord ischemia/reperfusion injury in rats.The abdominal aorta was blocked with a vascular clamp for 90 minutes and underwent reperfusion for 24 and 48 hours.The simple ischemia group and sham group served as controls.After rats had regained consciousness,hindlimbs showed varying degrees of functional impairment,and gradually improved with prolonged reperfusion in spinal cord ischemia/reperfusion injury groups.Hematoxylin-eosin staining demonstrated that neuronal injury and tissue edema were most severe in the 24-hour reperfusion group,and mitigated in the 48-hour reperfusion group.There were 8,242 differentially expressed m RNAs obtained by Multi-Class Dif in the simple ischemia group,24-hour and 48-hour reperfusion groups.Sixteen m RNA dynamic expression patterns were obtained by Serial Test Cluster.Of them,five patterns were significant.In the No.28 pattern,all differential genes were detected in the 24-hour reperfusion group,and their expressions showed a trend in up-regulation.No.11 pattern showed a decreasing trend in m RNA whereas No.40 pattern showed an increasing trend in m RNA from ischemia to 48 hours of reperfusion,and peaked at 48 hours.In the No.25 and No.27 patterns,differential expression appeared only in the 24-hour and 48-hour reperfusion groups.Among the five m RNA dynamic expression patterns,No.11 and No.40 patterns could distinguish normal spinal cord from pathological tissue.No.25 and No.27 patterns could distinguish simple ischemia from ischemia/reperfusion.No.28 pattern could analyze the need for inducing reperfusion injury.The study of specific pathways and functions for different dynamic patterns can provide a theoretical basis for clinical differential diagnosis and treatment of spinal cord ischemia/reperfusion injury.
基金the grants from Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, P. R. China (No. 2016LMFS-B02)the Key Research and Development Program of Shandong Province (No. 2016GSF115012)+1 种基金Basic Scientific Research Fund of YSFRI (No. 2060302201516054)Natural Science Foundation of Shandong Province (No. ZR2016 CQ32)
文摘The pen shell(Atrina pectinata) is a large wedge-shaped bivalve, which belongs to family Pinnidae. Due to its large and nutritious adductor muscle, it is the popular seafood with high commercial value in Asia-Pacific countries. However, limiting genomic and transcriptomic data have hampered its genetic investigations. In this study, the transcriptome of A. pectinata was deeply sequenced using Illumina pair-end sequencing technology. After assembling, a total of 127263 unigenes were obtained. Functional annotation indicated that the highest percentage of unigenes(18.60%) was annotated on GO database, followed by 18.44% on PFAM database and 17.04% on NR database. There were 270 biological pathways matched with those in KEGG database. Furthermore, a total of 23452 potential simple sequence repeats(SSRs) were identified, of them the most abundant type was mono-nucleotide repeats(12902, 55.01%), which was followed by di-nucleotide(8132, 34.68%), tri-nucleotide(2010, 8.57%), tetra-nucleotide(401, 1.71%), and penta-nucleotide(7, 0.03%) repeats. Sixty SSRs were selected for validating and developing genic SSR markers, of them 23 showed polymorphism in a cultured population with the average observed and expected heterozygosities of 0.412 and 0.579, respectively. In this study, we established the first comprehensive transcript dataset of A. pectinata genes. Our results demonstrated that RNA-Seq is a fast and cost-effective method for genic SSR development in non-model species.
基金supported by the National Natural Science Foundation of China(31301958)the Chinese Postdoctoral Science Foundation(2013T60808)
文摘High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA(long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix(Xist antisense RNA), chicken LXN(latexin) and GFM1(Gelongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.
基金supported by the National Key Research and De-velopment Program of China (2021YFC2103300),Research Equipment Program of Chinese Academy of Sciences (YJKYYQ20170023)National Natural Science Foundation of China (32071423,31470214,and 32200067)+1 种基金Natural Science Foundation of Hebei Province (C2020204013)Development Program Projects of Hebei Province (22322905D).
文摘Industrial manufacturing of bioproducts,especially bioethanol,can benefit from high-temperature fermentation,which requires the use of thermotolerant yeast strains.Mitochondrial activity in yeast is closely related to its over-all metabolism.However,the mitochondrial respiratory changes in response to adaptive thermotolerance are still poorly understood and have been rarely utilized for developing thermotolerant yeast cell factories.Here,adap-tive evolution and transcriptional sequencing,as well as whole-genome-level gene knockout,were used to obtain a thermotolerant strain of Saccharomyces cerevisiae.Furthermore,thermotolerance and bioethanol production efficiency of the engineered strain were examined.Physiological evaluation showed the boosted fermentation ca-pacity and suppressed mitochondrial respiratory activity in the thermotolerant strain.The improved fermentation produced an increased supply of adenosine triphosphate required for more active energy-consuming pathways.Transcriptome analysis revealed significant changes in the expression of the genes involved in the mitochondrial respiratory chain.Evaluation of mitochondria-associated gene knockout confirmed that ADK1,DOC1,or MET7 were the key factors for the adaptive evolution of thermotolerance in the engineered yeast strain.Intriguingly,overexpression of DOC1 with TEF1 promoter regulation led to a 10.1%increase in ethanol production at 42℃.The relationships between thermotolerance,mitochondrial activity,and respiration were explored,and a ther-motolerant yeast strain was developed by altering the expression of mitochondrial respiration-related genes.This study provides a better understanding on the physiological mechanism of adaptive evolution of thermotolerance in yeast.
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 suggested impaired antitumor immunity and increased metastatic ability during CRC progression.