The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sort...The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.展开更多
Microbiome-wide association studies(MWASs)have uncovered microbial markers linked to ecosystem traits,but the mechanisms underlying their functions can remain elusive.This is largely due to challenges in validating th...Microbiome-wide association studies(MWASs)have uncovered microbial markers linked to ecosystem traits,but the mechanisms underlying their functions can remain elusive.This is largely due to challenges in validating their in situ metabolic activities and tracing such activities to individual genomes.Here,we introduced a phylogeny-metabolism dual-directed singlecell genomics approach called fluorescence-in situ-hybridization-guided single-cell Raman-activated sorting and sequencing(FISH-scRACS-seq).It directly localizes individual cells from target taxon via an FISH probe for marker organism,profiles their in situ metabolic functions via single-cell Raman spectra,sorts cells of target taxonomy and target metabolism,and produces indexed,high-coverage,and precisely-one-cell genomes.From cyclohexane-contaminated seawater,cells representing the MWAS-derived marker taxon of g-Proteobacteria and that are actively degrading cyclohexane in situ were directly identified via FISH and Raman,respectively,then sorted and sequenced for one-cell full genomes.In such a Pseudoalteromonas fuliginea cell,we discovered a three-component cytochrome P450 system that can convert cyclohexane to cyclohexanol in vitro,representing a previously unknown group of cyclohexane-degrading enzymes and organisms.Therefore,by unveiling enzymes,pathways,genomes,and their in situ cellular functions specifically for those organisms with ecological relevance at one-cell resolution,FISH-scRACS-seq is a rational and generally applicable approach to dissecting and mining microbiota functions.展开更多
Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1...Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.展开更多
Due to the limitations of existing approaches,a rapid,sensitive,accurate,comprehensive,and generally applicable strategy to diagnose and treat bacterial and fungal infections remains a major challenge.Here,based on th...Due to the limitations of existing approaches,a rapid,sensitive,accurate,comprehensive,and generally applicable strategy to diagnose and treat bacterial and fungal infections remains a major challenge.Here,based on the ramanome technology platform,we propose a culture‐free,one cell resolution,phenome‐genome‐combined strategy called single‐cell identification,viability and vitality tests and source tracking(SCIVVS).For each cell directly extracted from a clinical specimen,the fingerprint region of the D2O‐probed single cell Raman spectrum(SCRS)enables species‐level identification based on a reference SCRS database of pathogen species,whereas the C‐D band accurately quantifies viability,metabolic vitality,phenotypic susceptibility to antimicrobials,and their intercellular heterogeneity.Moreover,to source track a cell,Raman‐activated cell sorting followed by sequencing or cultivation proceeds,producinging an indexed,high coverage genome assembly or a pure culture from precisely one pathogenic cell.Finally,an integrated SCIVVS workflow that features automated profiling and sorting of metabolic and morphological phenomes can complete the entire process in only a few hours.Because it resolves heterogeneity for both the metabolic phenome and genome,targets functions,can be automated,and is orders‐of‐magnitude faster while cost‐effective,SCIVVS is a new technological and data framework to diagnose and treat bacterial and fungal infections in various clinical and disease control settings.展开更多
基金funded by National Key Research and Development Program of China(grant number 2022YFC3502002)CAS-NSTDA Joint Research Project(2024)(grant number 101GJHZ2024017MI).
文摘The current single-cell analysis technologies such as fluorescence-activated cell sorting(FACS)and fluorescence-activated droplet sorting(FADS)could decipher the cellular heterogeneity but were constrained by low sorting performance and cell viability.Here,an ultra-sensitive single-cell sorting platform has been developed by integrating the FADS technology with Tetramer-HCR-EvaGreen(THE)fluorescence signal amplification.The THE system produced much higher fluorescence signal than that of the single Tetramer or Tetramer-HCR signal amplification.Upon application to target MCF-7 cells,the platform exhibited high efficacy and selectivity while maintaining more than 95%cell viability.The THE-FADS achieved sorting efficiencies of 55.5%and 50.3%with purities of 91%and 85%for MCF-7 cells in PBS solutions and simulated serum samples,respectively.The sorted MCF-7 cells showed similar proliferation together with CK19 and EGFR mRNA expression compared with the control cells.The established THE-FADS showed the promising prospects to cellular heterogeneity understanding and personalized medicine.
基金supported by grants from the National Key R&D Program Young Scientists Project of China(2021YFD1900400)the Natural Science Foundation of China(32030003,32270109,32370097,42076165,and 32071266).
文摘Microbiome-wide association studies(MWASs)have uncovered microbial markers linked to ecosystem traits,but the mechanisms underlying their functions can remain elusive.This is largely due to challenges in validating their in situ metabolic activities and tracing such activities to individual genomes.Here,we introduced a phylogeny-metabolism dual-directed singlecell genomics approach called fluorescence-in situ-hybridization-guided single-cell Raman-activated sorting and sequencing(FISH-scRACS-seq).It directly localizes individual cells from target taxon via an FISH probe for marker organism,profiles their in situ metabolic functions via single-cell Raman spectra,sorts cells of target taxonomy and target metabolism,and produces indexed,high-coverage,and precisely-one-cell genomes.From cyclohexane-contaminated seawater,cells representing the MWAS-derived marker taxon of g-Proteobacteria and that are actively degrading cyclohexane in situ were directly identified via FISH and Raman,respectively,then sorted and sequenced for one-cell full genomes.In such a Pseudoalteromonas fuliginea cell,we discovered a three-component cytochrome P450 system that can convert cyclohexane to cyclohexanol in vitro,representing a previously unknown group of cyclohexane-degrading enzymes and organisms.Therefore,by unveiling enzymes,pathways,genomes,and their in situ cellular functions specifically for those organisms with ecological relevance at one-cell resolution,FISH-scRACS-seq is a rational and generally applicable approach to dissecting and mining microbiota functions.
基金the National Natural Science Foundation of China(82020108021 and 81530063)the Projects of the State Key Laboratory of Trauma,Burns and Combined Injury(SKLYQ201901)+1 种基金the Chongqing Science and Technology Talents,Chongqing Special Project for Academicians(cstc2020yszx-jcyjX0004)the Training Plan for Innovation Ability on the Frontiers of Military Medical Research(2019CXJSB014).
文摘Purpose:The incidence of acute lung injury(ALI)in severe trauma patients is 48%and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%.Alveolar epithelial type 1 cells(AEC1 s)and type 2 cells(AEC2s)are the key cells in the repair of injured lungs as well as fetal lung development.Therefore,the purification and culture of AECls and AEC2s play an important role in the research of repair and regeneration of lung tissue.Methods:Sprague-Dawley rats(3-4 weeks,120-150 g)were purchased for experiment.Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells,and then magnetic bead cell sorting was performed to isolate Tla positive cells as AECls from the single-cell suspension by using polyclonal rabbit anti-Tla(a specific AECls membrane protein)antibodies combined with anti-rabbit IgG microbeads.Afterwards,alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining Tla-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads.Cell purity was identified by immunofluorescence staining and flow cytometry.Resii沾••The purity of AECls and AEC2s was 88.3%±3.8%and 92.6%±2.7%,respectively.The cell growth was observed as follows:AECls stretched within the 12-16 h,but the cells proliferated slowly;while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.Conclusion:AECls and AEC2s sorted by this method have high purity and good viability.Therefore,our method provides a new approach for the isolation and culture of AECls and AEC2s as well as a new strategy for the research of lung repair and regeneration.
基金National Key R&D Program of China,Grant/Award Number:2022YFA1304101CAS,Grant/Award Number:XDB29050400+1 种基金National Natural Science Foundation of China,Grant/Award Number:32030003Shenzhen‐Hong Kong Innovation Circle Plan,Grant/Award Number:SGDX2019081623060946。
文摘Due to the limitations of existing approaches,a rapid,sensitive,accurate,comprehensive,and generally applicable strategy to diagnose and treat bacterial and fungal infections remains a major challenge.Here,based on the ramanome technology platform,we propose a culture‐free,one cell resolution,phenome‐genome‐combined strategy called single‐cell identification,viability and vitality tests and source tracking(SCIVVS).For each cell directly extracted from a clinical specimen,the fingerprint region of the D2O‐probed single cell Raman spectrum(SCRS)enables species‐level identification based on a reference SCRS database of pathogen species,whereas the C‐D band accurately quantifies viability,metabolic vitality,phenotypic susceptibility to antimicrobials,and their intercellular heterogeneity.Moreover,to source track a cell,Raman‐activated cell sorting followed by sequencing or cultivation proceeds,producinging an indexed,high coverage genome assembly or a pure culture from precisely one pathogenic cell.Finally,an integrated SCIVVS workflow that features automated profiling and sorting of metabolic and morphological phenomes can complete the entire process in only a few hours.Because it resolves heterogeneity for both the metabolic phenome and genome,targets functions,can be automated,and is orders‐of‐magnitude faster while cost‐effective,SCIVVS is a new technological and data framework to diagnose and treat bacterial and fungal infections in various clinical and disease control settings.
