Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tool...Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging.This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp.The destination vector sets contained two Gateway cassettes flanked by Minos sites,allowing the N-or C-terminal tagging of a protein of interest with various fluorescent markers.In addition,we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry,a fluorescent indicator for Cas9 expression at cellular resolution.We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs.Furthermore,we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues.The genetic tools developed in this study offer a robust method for gene expression,cell tracking,and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.展开更多
Regulation of microRNAs(miRNAs)on various biological processes has been a surprising and exciting field.Identification of miRNAs is the first step to comprehensively understand their functions.However,attempts on glob...Regulation of microRNAs(miRNAs)on various biological processes has been a surprising and exciting field.Identification of miRNAs is the first step to comprehensively understand their functions.However,attempts on global identification and functional verification of miRNAs are very limited in penaeid shrimp Marsupenaeus japonicus,an economically important aquatic species.By performing an integrated analysis of transcriptomic profile from gastrula embryos of M.japonicus,21conserved miRNAs in M.japonicas(mja-miRNAs),belonging to 19 miRNA families,were identified and characterized.Of the 21 mja-miRNAs,15 miRNAs were successfully verified to be predominantly expressed in gastrula stage,where they displayed dynamic expression patterns compared with those in naupliuin stage.Based on perfect or near-perfect match to target region,120 target genes were predicted at transcriptome-wide level.Noteworthy,gene ontology(GO)classification and metabolic pathway annotation revealed eight targets that were actively involved in developmental processes.Of the predicted miRNA-mRNA pairs,six targets were then randomly selected and experimentally validated by dual luciferase reporter assay,where three pairs were proved with potential targeting activity.Overall,to search for conserved miRNAs potentially involved in early development of M.japonicus,we combined in silico and experimental methods,which can be applied in other organisms as well.Our data implied important roles of miRNAs in the early embryonic development and also suggested the presence of complex miRNA-mRNA functional networks in M.japonicus.展开更多
This paper delves into the lateral load-bearing behavior of lattice-shaped diaphragm wall(LSDW),a novel type of diaphragm wall foundation with many engineering advantages.By employing a double-layer wall structure for...This paper delves into the lateral load-bearing behavior of lattice-shaped diaphragm wall(LSDW),a novel type of diaphragm wall foundation with many engineering advantages.By employing a double-layer wall structure for the first time in laboratory settings,the research presents an innovative testing methodology,complete with novel computational formulas,to accurately measure the responses of LSDW’s inner and outer walls under varying loads.It is found that the Q-s curves of LSDWs exhibit a continuous,progressive deformation and failure characteristic without any abrupt drops,and the standard for judging the horizontal bearing capacity of LSDW foundations should be based on the allowable displacement of the superstructure.The bearing capacity for the double-chamber LSDWs was found to be approximately 1.68 times that of the single-chamber structure,pointing to a complex interplay between chamber number and structural capacity that extends beyond a linear relationship and incorporates the group wall effect.The study also reveals that LSDWs act as rigid bodies with minimal angular displacement and a consistent tilting deformation,peaking in bending moment at about 0.87 of wall depth from the mud surface,across different chamber configurations.Furthermore,it can be found that using the p-y curve method for analyzing the horizontal behavior of LSDW foundations is feasible,and the hyperbolic p-y curve method offers higher accuracy in calculations.These insights offer valuable guidance for both field and laboratory testing of LSDWs and aid in the design and calculation of foundations under horizontal loads.展开更多
基金supported by the National Key Research and Development Program of China(2022YFC2601302)the Taishan Scholar Program of Shan-dong Province,China(B.D.).
文摘Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging.This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp.The destination vector sets contained two Gateway cassettes flanked by Minos sites,allowing the N-or C-terminal tagging of a protein of interest with various fluorescent markers.In addition,we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry,a fluorescent indicator for Cas9 expression at cellular resolution.We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs.Furthermore,we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues.The genetic tools developed in this study offer a robust method for gene expression,cell tracking,and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.
基金the National Natural Science Foundation of China(No.32273116)the Natural Science Foundation of Shandong Province(China)(No.ZR2020MC189)the National Key R&D Program of China(No.2018YFD0901301)。
文摘Regulation of microRNAs(miRNAs)on various biological processes has been a surprising and exciting field.Identification of miRNAs is the first step to comprehensively understand their functions.However,attempts on global identification and functional verification of miRNAs are very limited in penaeid shrimp Marsupenaeus japonicus,an economically important aquatic species.By performing an integrated analysis of transcriptomic profile from gastrula embryos of M.japonicus,21conserved miRNAs in M.japonicas(mja-miRNAs),belonging to 19 miRNA families,were identified and characterized.Of the 21 mja-miRNAs,15 miRNAs were successfully verified to be predominantly expressed in gastrula stage,where they displayed dynamic expression patterns compared with those in naupliuin stage.Based on perfect or near-perfect match to target region,120 target genes were predicted at transcriptome-wide level.Noteworthy,gene ontology(GO)classification and metabolic pathway annotation revealed eight targets that were actively involved in developmental processes.Of the predicted miRNA-mRNA pairs,six targets were then randomly selected and experimentally validated by dual luciferase reporter assay,where three pairs were proved with potential targeting activity.Overall,to search for conserved miRNAs potentially involved in early development of M.japonicus,we combined in silico and experimental methods,which can be applied in other organisms as well.Our data implied important roles of miRNAs in the early embryonic development and also suggested the presence of complex miRNA-mRNA functional networks in M.japonicus.
基金supported by the National Nature Science Foundation of China(Grant No.42007247)the National Foreign Expert Project of China(No.DL2023036001L).
文摘This paper delves into the lateral load-bearing behavior of lattice-shaped diaphragm wall(LSDW),a novel type of diaphragm wall foundation with many engineering advantages.By employing a double-layer wall structure for the first time in laboratory settings,the research presents an innovative testing methodology,complete with novel computational formulas,to accurately measure the responses of LSDW’s inner and outer walls under varying loads.It is found that the Q-s curves of LSDWs exhibit a continuous,progressive deformation and failure characteristic without any abrupt drops,and the standard for judging the horizontal bearing capacity of LSDW foundations should be based on the allowable displacement of the superstructure.The bearing capacity for the double-chamber LSDWs was found to be approximately 1.68 times that of the single-chamber structure,pointing to a complex interplay between chamber number and structural capacity that extends beyond a linear relationship and incorporates the group wall effect.The study also reveals that LSDWs act as rigid bodies with minimal angular displacement and a consistent tilting deformation,peaking in bending moment at about 0.87 of wall depth from the mud surface,across different chamber configurations.Furthermore,it can be found that using the p-y curve method for analyzing the horizontal behavior of LSDW foundations is feasible,and the hyperbolic p-y curve method offers higher accuracy in calculations.These insights offer valuable guidance for both field and laboratory testing of LSDWs and aid in the design and calculation of foundations under horizontal loads.
