Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains chal...Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.展开更多
The interrelationships and synergistic regulations of bioactive molecules play pivotal roles in physiological and pathological processes involved in the initiation and development of some diseases,such as cancer and n...The interrelationships and synergistic regulations of bioactive molecules play pivotal roles in physiological and pathological processes involved in the initiation and development of some diseases,such as cancer and neurodegenerative and cardiovascular diseases.Therefore,the simultaneous,accurate and timely detection of two bioactive molecules is crucial to explore their roles and pathological mechanisms in related diseases.Fluorescence imaging associated with small molecular probes has been widely used in the imaging of bioactive molecules in living cells and in vivo due to its excellent performances,including high sensitivity and selectivity,noninvasive properties,real-time and high spatial temporal resolution.Single organic molecule fluorescent probes have been successively developed to simultaneously monitor two biomolecules to uncover their synergistic relationships in living systems.Hence,in this review,we focus on summarizing the design strategies,classifications,and bioimaging applications of dual-response fluorescent probes over the past decade.Furthermore,future research directions in this field are proposed.展开更多
In recent years,the shortage of available organs for transplant patients has grown exponentially across the globe.Consequently,the healthcare industry is in dire need of artificial substitutes.Many recent research stu...In recent years,the shortage of available organs for transplant patients has grown exponentially across the globe.Consequently,the healthcare industry is in dire need of artificial substitutes.Many recent research studies and tissue engineering groups have decided to utilize 3D bioprinting to produce these artificial organs.This synthetic organ printing is made possible by advancements in the materials required for the constructs,the printing method-ologies used to produce them,and the final physical structures’varying properties.The cutting-edge research and technology related to 3D and 4D live cell bioprinting have recently allowed researchers to produce multiple types of artificial organs and tissues.These tissues can be utilized for drug screening and organ replacement applica-tions.This article provides an extensive review of all the pertinent 3D live cell bioprinting technologies.First,we describe scaffolding methods and their comparison with the traditional technologies.Second,we explain the 3D bioprinting technology,its evolution,and its multiple types.Moreover,we describe the pros and cons of each bioprinting method.Third,we have discussed the critical bioink properties and their impact on the formation of 3D bioprinting models.In addition,we also describe the mechanical properties of bioprinters.Fourth,we have thoroughly discussed the various types of hydrogels and their properties.Every kind of hydrogel is utilized in specific applications,and we have presented a comprehensive list of its advantages and disadvantages.Fifth,we have discussed various applications of 3D bioprinting technology.We have considered a case study of human or-gans and elaborated on how bioprinters can revolutionize the organ replacement industry.Finally,we evaluated the possibility of 4D printing in the future organ industry,incorporating temporal factors into the bioprinting process.展开更多
The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction.CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivate...The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction.CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9)labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However,the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module)and scFv-sfGFP-NLS(signal module).We removed the nuclear location sequence(NLS)of the signal module and inserted two copies of EGFP into the signal module.The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio)was decreased by 71%,and the ratio of the signal to the background(S/B ratio)was increased by 1.6 times.The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.展开更多
Cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular functionalities.This proces...Cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular functionalities.This process is integral to diverse biological phenomena,including embryonic development,cell migration,tissue regeneration,and disease pathology,particularly in the context of cancer metastasis and cardiovascular diseases.Despite the profound biological and clinical significance of mechanotransduction,our understanding of this complex process remains incomplete.The recent development of advanced optical techniques enables in-situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside a cell.In this review,we delved into the current state-of-the-art techniques utilized to probe cellular mechanobiology,their principles,applications,and limitations.We mainly examined optical methodologies to quantitatively measure the mechanical properties of cells during intracellular transport,cell adhesion,and migration.We provided an introductory overview of various conventional and optical-based techniques for probing cellular mechanics.These techniques have provided into the dynamics of mechanobiology,their potential to unravel mechanistic intricacies and implications for therapeutic intervention.展开更多
Fe-based single-atomic site catalysts(SASCs),with the natural metalloproteases-like active site structure,have attracted widespread attention in biocatalysis and biosensing.Precisely,controlling the isolated single-at...Fe-based single-atomic site catalysts(SASCs),with the natural metalloproteases-like active site structure,have attracted widespread attention in biocatalysis and biosensing.Precisely,controlling the isolated single-atom Fe-N-C active site structure is crucial to improve the SASCs’performance.In this work,we use a facile ion-imprinting method(IIM)to synthesize isolated Fe-N-C single-atomic site catalysts(IIM-Fe-SASC).With this method,the ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites.The IIM-Fe-SASC shows better peroxidase-like activities than that of non-imprinted references.Due to its excellent properties,IIM-Fe-SASC is an ideal nanoprobe used in the colorimetric biosensing of hydrogen peroxide(H_(2)O_(2)).Using IIM-Fe-SASC as the nanoprobe,in situ detection of H_(2)O_(2)generated from MDA-MB-231 cells has been successfully demonstrated with satisfactory sensitivity and specificity.This work opens a novel and easy route in designing advanced SASC and provides a sensitive tool for intracellular H_(2)O_(2)detection.展开更多
Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for t...Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or展开更多
The semiconductive characteristics of clectron-transfrring proteins in living cells E coli was investigated by electrochemsical impedance spectroscopy(EIS). We found that the electrochemical impedance of living cells ...The semiconductive characteristics of clectron-transfrring proteins in living cells E coli was investigated by electrochemsical impedance spectroscopy(EIS). We found that the electrochemical impedance of living cells as a function of temprature followed the Arrhenius equation for semiconductors. This result shows a strong evidence to prove the semiconductive behavior of proteins展开更多
In the past few decades,robotics research has witnessed an increasingly high interest in miniaturized,intelligent,and integrated robots.The imperative component of a robot is the actuator that determines its performan...In the past few decades,robotics research has witnessed an increasingly high interest in miniaturized,intelligent,and integrated robots.The imperative component of a robot is the actuator that determines its performance.Although traditional rigid drives such as motors and gas engines have shown great prevalence in most macroscale circumstances,the reduction of these drives to the millimeter or even lower scale results in a significant increase in manufacturing difficulty accompanied by a remarkable performance decline.Biohybrid robots driven by living cells can be a potential solution to overcome these drawbacks by benefiting from the intrinsic microscale self-assembly of living tissues and high energy efficiency,which,among other unprecedented properties,also feature flexibility,self-repair,and even multiple degrees of freedom.This paper systematically reviews the development of biohybrid robots.First,the development of biological flexible drivers is introduced while emphasizing on their advantages over traditional drivers.Second,up-to-date works regarding biohybrid robots are reviewed in detail from three aspects:biological driving sources,actuator materials,and structures with associated control methodologies.Finally,the potential future applications and major challenges of biohybrid robots are explored.展开更多
A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstr...A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.展开更多
The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles(GNPs) based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNP...The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles(GNPs) based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM) and classic flow cytometry(FCM) assay, and satisfactory results were obtained.展开更多
Exact interaction mechanism between Bax and Bcl-XL,two key Bcl-2 family proteins,is an interesting and controversial issue.Partial acceptor photobleaching-based quantitative fluores-cence resonance energy transfer(FRE...Exact interaction mechanism between Bax and Bcl-XL,two key Bcl-2 family proteins,is an interesting and controversial issue.Partial acceptor photobleaching-based quantitative fluores-cence resonance energy transfer(FRET)measurement,PbFRET,is a widely used FRET quantification method in living cells.In this report,we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency(E)images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL.The E value between CFP-Bax and YFP-Bcl-XL was 4.59%in cytosol and 11.31%on mitochondria,conclusively indicating the direct interaction of the two proteins,and the interaction of the two proteins was strong on mitochondria and modest in cytosol.展开更多
Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used ...Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.展开更多
Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin...Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells (SUVEC). The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein (EGFP) fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase (15% to 20% increase) and fewer were in S phase (18% decrease) compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.展开更多
Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for b...Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.展开更多
Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficie...Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest(ROI)function of confocal microscope and partial acceptor photobleaching.We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3,and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine(STS)-induced apoptosis.Our results for thefirst demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.展开更多
Cellular micro-irradiation is now recognized as a powerful technique to unveil the mechanisms of interaction between ionizing radiation and living cells or tissues. The single-ion microbeam (SIM) is uniquely capable...Cellular micro-irradiation is now recognized as a powerful technique to unveil the mechanisms of interaction between ionizing radiation and living cells or tissues. The single-ion microbeam (SIM) is uniquely capable of delivering precisely the predefined number of charged particles (precise doses of radiation) to specific individual cells or sub-cellular targets in situ. No active research in the field concerning the original process of the interaction between low-energy ions and complicated organisms has been reported yet. To address this challenge, the aim of the present design is to further wrestle with multi-dimensional quantitative information from living cells traversed by an exact number of ions real-time rather than endpoints, in the time scale from milliseconds to days.展开更多
Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a...Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.展开更多
Dear Editor,CRISPR-Cas9 (clustered regularly interspaced short palin- dromic repeats-CRISPR associated) systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation,...Dear Editor,CRISPR-Cas9 (clustered regularly interspaced short palin- dromic repeats-CRISPR associated) systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation, and chromosome loci imaging (Dominguez et al., 2016; Komor et al., 2017). A typical engineered CRISPR-Cas9 system is composed of a Cas9 protein and a single guide RNA (sgRNA), which could form a protein/RNA complex to recognize and cleave DNA sequence (Hsu et al., 2014; Wright et al., 2016).展开更多
基金supported by the following grants:National Natural Science Foundation of China(62125504,62361166631)STI 2030-Major Projects(2021ZD0200401)+1 种基金the Fundamental Research Funds for the Central Universities(226-2022-00201)the Open Project Program of Wuhan National Laboratory for Optoelectronics(2021WNLOKF007).
文摘Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.
基金supported by the National Natural Science Foundation of China(Grant Nos.91753111,22074083 and 21927811)the Key Research and Development Program of Shandong Province(Grant No.2018YFJH0502)the National Major Scientific and Technological Special Project for Significant New Drugs Development(Grant No.2017ZX09301030004).
文摘The interrelationships and synergistic regulations of bioactive molecules play pivotal roles in physiological and pathological processes involved in the initiation and development of some diseases,such as cancer and neurodegenerative and cardiovascular diseases.Therefore,the simultaneous,accurate and timely detection of two bioactive molecules is crucial to explore their roles and pathological mechanisms in related diseases.Fluorescence imaging associated with small molecular probes has been widely used in the imaging of bioactive molecules in living cells and in vivo due to its excellent performances,including high sensitivity and selectivity,noninvasive properties,real-time and high spatial temporal resolution.Single organic molecule fluorescent probes have been successively developed to simultaneously monitor two biomolecules to uncover their synergistic relationships in living systems.Hence,in this review,we focus on summarizing the design strategies,classifications,and bioimaging applications of dual-response fluorescent probes over the past decade.Furthermore,future research directions in this field are proposed.
文摘In recent years,the shortage of available organs for transplant patients has grown exponentially across the globe.Consequently,the healthcare industry is in dire need of artificial substitutes.Many recent research studies and tissue engineering groups have decided to utilize 3D bioprinting to produce these artificial organs.This synthetic organ printing is made possible by advancements in the materials required for the constructs,the printing method-ologies used to produce them,and the final physical structures’varying properties.The cutting-edge research and technology related to 3D and 4D live cell bioprinting have recently allowed researchers to produce multiple types of artificial organs and tissues.These tissues can be utilized for drug screening and organ replacement applica-tions.This article provides an extensive review of all the pertinent 3D live cell bioprinting technologies.First,we describe scaffolding methods and their comparison with the traditional technologies.Second,we explain the 3D bioprinting technology,its evolution,and its multiple types.Moreover,we describe the pros and cons of each bioprinting method.Third,we have discussed the critical bioink properties and their impact on the formation of 3D bioprinting models.In addition,we also describe the mechanical properties of bioprinters.Fourth,we have thoroughly discussed the various types of hydrogels and their properties.Every kind of hydrogel is utilized in specific applications,and we have presented a comprehensive list of its advantages and disadvantages.Fifth,we have discussed various applications of 3D bioprinting technology.We have considered a case study of human or-gans and elaborated on how bioprinters can revolutionize the organ replacement industry.Finally,we evaluated the possibility of 4D printing in the future organ industry,incorporating temporal factors into the bioprinting process.
基金the National Natural Science Foundation of China(21890743)Strategic Priority Research Program of the Chinese Academy of Sciences,China(XDB29050100)National Key Research and Development Program of China(2017YFA0205503)。
文摘The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction.CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9)labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However,the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module)and scFv-sfGFP-NLS(signal module).We removed the nuclear location sequence(NLS)of the signal module and inserted two copies of EGFP into the signal module.The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio)was decreased by 71%,and the ratio of the signal to the background(S/B ratio)was increased by 1.6 times.The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.
基金the funding from Start-up Fundings of Ocean University of China(862401013154 and 862401013155)Laboratory for Marine Drugs and Bioproducts Qingdao Marine Science and Technology Center(LMDBCXRC202401 and LMDBCXRC202402)+1 种基金Taishan Scholar Youth Expert Program of Shandong Province(tsqn202306102 and tsqn202312105)Shandong Provincial Overseas Excellent Young Scholar Program(2024HWYQ-042 and 2024HWYQ-043)for supporting this work.
文摘Cellular mechanotransduction characterized by the transformation of mechanical stimuli into biochemical signals,represents a pivotal and complex process underpinning a multitude of cellular functionalities.This process is integral to diverse biological phenomena,including embryonic development,cell migration,tissue regeneration,and disease pathology,particularly in the context of cancer metastasis and cardiovascular diseases.Despite the profound biological and clinical significance of mechanotransduction,our understanding of this complex process remains incomplete.The recent development of advanced optical techniques enables in-situ force measurement and subcellular manipulation from the outer cell membrane to the organelles inside a cell.In this review,we delved into the current state-of-the-art techniques utilized to probe cellular mechanobiology,their principles,applications,and limitations.We mainly examined optical methodologies to quantitatively measure the mechanical properties of cells during intracellular transport,cell adhesion,and migration.We provided an introductory overview of various conventional and optical-based techniques for probing cellular mechanics.These techniques have provided into the dynamics of mechanobiology,their potential to unravel mechanistic intricacies and implications for therapeutic intervention.
基金This work was supported by a WSU startup fund.XAS measurements were done at beamline 12-BM of the Advanced Photon Source(APS),which is a User Facility operated for the U.S.Department of Energy Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357.
文摘Fe-based single-atomic site catalysts(SASCs),with the natural metalloproteases-like active site structure,have attracted widespread attention in biocatalysis and biosensing.Precisely,controlling the isolated single-atom Fe-N-C active site structure is crucial to improve the SASCs’performance.In this work,we use a facile ion-imprinting method(IIM)to synthesize isolated Fe-N-C single-atomic site catalysts(IIM-Fe-SASC).With this method,the ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites.The IIM-Fe-SASC shows better peroxidase-like activities than that of non-imprinted references.Due to its excellent properties,IIM-Fe-SASC is an ideal nanoprobe used in the colorimetric biosensing of hydrogen peroxide(H_(2)O_(2)).Using IIM-Fe-SASC as the nanoprobe,in situ detection of H_(2)O_(2)generated from MDA-MB-231 cells has been successfully demonstrated with satisfactory sensitivity and specificity.This work opens a novel and easy route in designing advanced SASC and provides a sensitive tool for intracellular H_(2)O_(2)detection.
基金supported by US National Institutes of Health grant R01 AI44902 (to C Z )a Pre-doctoral Fellowship from the American Heart Association (to W C )
文摘Introduction Integrins are a large family of adhesion molecules broadly expressed on the surface of a wide variety of cells as heterodimers. Binding of integrins to ligands provides not only mechanical anchorage for the cell to another cell or
文摘The semiconductive characteristics of clectron-transfrring proteins in living cells E coli was investigated by electrochemsical impedance spectroscopy(EIS). We found that the electrochemical impedance of living cells as a function of temprature followed the Arrhenius equation for semiconductors. This result shows a strong evidence to prove the semiconductive behavior of proteins
基金the Research Project Funding of National University of Defense Technology of China(No.ZK19-33)the National Postdoctoral International Exchange Program Funding for Incoming Postdoctoral Students(postdoctoral No.48127).
文摘In the past few decades,robotics research has witnessed an increasingly high interest in miniaturized,intelligent,and integrated robots.The imperative component of a robot is the actuator that determines its performance.Although traditional rigid drives such as motors and gas engines have shown great prevalence in most macroscale circumstances,the reduction of these drives to the millimeter or even lower scale results in a significant increase in manufacturing difficulty accompanied by a remarkable performance decline.Biohybrid robots driven by living cells can be a potential solution to overcome these drawbacks by benefiting from the intrinsic microscale self-assembly of living tissues and high energy efficiency,which,among other unprecedented properties,also feature flexibility,self-repair,and even multiple degrees of freedom.This paper systematically reviews the development of biohybrid robots.First,the development of biological flexible drivers is introduced while emphasizing on their advantages over traditional drivers.Second,up-to-date works regarding biohybrid robots are reviewed in detail from three aspects:biological driving sources,actuator materials,and structures with associated control methodologies.Finally,the potential future applications and major challenges of biohybrid robots are explored.
文摘A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.
基金Supported by the National Natural Science Foundation of China(No.20875087)the Fund of Chinese Academy of Sciences (No.KJCX2-YW-H11)
文摘The interactions of antibiotic with living cells were studied by lectin conjugated gold nanoparticles(GNPs) based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay. In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM) and classic flow cytometry(FCM) assay, and satisfactory results were obtained.
基金supported by the National Natural Science Foundation of China(NSFC)(Grant Nos.61527825,61875056 and 81572184)
文摘Exact interaction mechanism between Bax and Bcl-XL,two key Bcl-2 family proteins,is an interesting and controversial issue.Partial acceptor photobleaching-based quantitative fluores-cence resonance energy transfer(FRET)measurement,PbFRET,is a widely used FRET quantification method in living cells.In this report,we implemented pixel-to-pixel PbFRET imaging on a wide-field microscope to map the FRET efficiency(E)images of single living HepG2 cells co-expressing CFP-Bax and YFP-Bcl-XL.The E value between CFP-Bax and YFP-Bcl-XL was 4.59%in cytosol and 11.31%on mitochondria,conclusively indicating the direct interaction of the two proteins,and the interaction of the two proteins was strong on mitochondria and modest in cytosol.
基金supported in part by the National Natural Science Foundation of China(61871251 and 61871022)Sichuan Science and Technology Program(2019YFSY0048)
文摘Intensity-based quantitative fluorescence resonance energy transfer(FRET)is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit.This widely used technique needs complicated experimental process and manual image analyses to obtain precise results,which take a long time and restrict the application of quantitative FRET especially in living cells.In this paper,a simplified and automatic quanti-tative FRET(saqFRET)method with high efficiency is presented.In saqFRET,photo-activatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein(EGFP)are used to simplify FRET crosstalk elimination.Traditional manual image analyses are time consuming when the dataset is large.The proposed automatic image analyses based on deep learning can analyze 100 samples within 30 s and demonstrate the same precision as manual image analyses.
文摘Cyclin A is a key regulator of the cell cycle. Its expression may become disrupted in virusinfected cells, leading to deregulation of the cell cycle and increased cell proliferation. Here, we cloned the porcine cyclin A gene and verified its functionality in swine umbilicus vein endothelial cells (SUVEC). The human cyclin A gene was used to probe databases to clone the pig cyclin A gene electronically. The identified porcine cDNA contained an open reading frame of 1,299 bp, encoding 432 amino acids, the same length as the human cyclin A protein. The porcine cyclin A gene comprises eight exons on chromosome 8. The sequence of the in silico clone and expression of this novel gene were confirmed in SUVEC by reverse transcription PCR. Western blotting of cell lysates from SUVEC transfected with a cyclin A enhanced green fluorescent protein (EGFP) fusion construct revealed a band at approximately 40 kDa. Confocal microscopy of CycA-EGFP-expressing cells showed that the fusion protein was expressed in the nucleus. Flow cytometry demonstrated that more stably expressing SUVEC-CycA-EGFP were in G1 phase (15% to 20% increase) and fewer were in S phase (18% decrease) compared with control ceils. MTS assays showed that the proliferative activity of SUVEC-Cy- cAG-EGFP was significantly higher than that of the control cells. In conclusion, we have cloned the pig cyclin A gene and demonstrated that its biological function is consistent with cyclin A in other mammali- an species. This provides a foundation for future research on the impact of virus infection on cyclin A.
基金supported by the National Key R&D Program of China(2018YFC0910600)the National Natural Science Foundation of China(61605124/41603059/61525503/61620106016/61835009/81727804)+3 种基金Project of Department of Education of Guangdong Province(2015KGJHZ002/2016KCXTD007)Guangdong Natural Science Foundation Innovation Team(2014A030312008)Shenzhen Basic Research Project(JCYJ20150930104948169/JCYJ20160328144746940/JCYJ20170412105003520)the Natural Science Foundation of Shenzhen University(2019108).
文摘Inorganic quantum dots(QDs)have excellent optical properties,such as high°uorescence intensity,excellent photostability and tunable emission wavelength,etc.,facilitating them to be used as labels and probes for bioimaging.In this study,CdSe@ZnS QDs are used as probes for Fluorescence lifetime imaging microscope(FLIM)and stimulated emission depletion(STED)nanoscopy imaging.The emission peak of CdSe@ZnS QDs centered at 526 nm with a narrow width of 19 nm and the photoluminescence quantum yield(PLQY)was 64%.The QDs presented excellent anti-photobleaching property which can be irradiated for 400 min by STED laser with 39.8 mW.The lateral resolution of 42.0 nm is demonstrated for single QDs under STED laser(27.5 mW)irradiation.Furthermore,the CdSe@ZnS QDs were for the first time used to successfully label the lysosomes of living HeLa cells and 81.5 nm lateral resolution is obtained indicating the available super-resolution applications in living cells for inorganic QD probes.Meanwhile,Eca-109 cells labeled with the CdSe@ZnS QDs was observed with FLIM,and their fluorescence lifetime was around 3.1 ns,consistent with the in vitro value,suggesting that the QDs could act as a satisfactory probe in further FLIM-STED experiments.
基金This work is supported by the National Natural Science Foundation of China(NSFC)(Grant 81071491)Key Project of the Department of Education and Finance of Guangdong Province(cxzd115).
文摘Fluorescence resonance energy transfer(FRET)technology had been widely used to study proteinprotein interactions in living cells.In this study,we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest(ROI)function of confocal microscope and partial acceptor photobleaching.We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3,and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine(STS)-induced apoptosis.Our results for thefirst demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.
基金the National Major Technologies R&D Program of China during the 10tb Five-Year Plan Period(No.2001BA30213)the National Science Fund for Distinguished Young Scholars(No.10225526)+1 种基金the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KSCX2-SW-324)the Foundation for Key University Teachers by the Ministry of Education(No.2005jq1135)
文摘Cellular micro-irradiation is now recognized as a powerful technique to unveil the mechanisms of interaction between ionizing radiation and living cells or tissues. The single-ion microbeam (SIM) is uniquely capable of delivering precisely the predefined number of charged particles (precise doses of radiation) to specific individual cells or sub-cellular targets in situ. No active research in the field concerning the original process of the interaction between low-energy ions and complicated organisms has been reported yet. To address this challenge, the aim of the present design is to further wrestle with multi-dimensional quantitative information from living cells traversed by an exact number of ions real-time rather than endpoints, in the time scale from milliseconds to days.
基金supported by the Natural Science Foundation of China(Nos.21877030,21735002,21778016 and 21521063)。
文摘Accurate detection and imaging of adenosine triphosphate(ATP)expression levels in living cells is of great value for understanding cell metabolism,physiological activities,and pathologic mechanisms.Here,we developed a DNA tetrahedron-based split aptamer probe(TD probe)for ratiometric fluorescence imaging of ATP in living cells.The TD probe is constructed by hybridizing two split ATP aptamer probes(Apt-a and Apt-b)to a DNA tetrahedron assembled by four DNA oligonucleotides(T1,T2,T3 and T4).In the presence of ATP,the TD probe will alter its structure from the open to closed state,thus bringing the separated donor and acceptor fluorophores into close proximity for high fluorescence resonance energy transfer(FRET)signals.The TD probe exhibits low cytotoxicity,efficient cell internalization and good biological stability.Moreover,based on the FRET“off”to“on”signal output mode,the TD probe can effectively avoid false-positive signals from complex biological matrices,which is significant for long-term reliable imaging in living cells.In addition,by changing the split aptamers attached to DNA tetrahedron,the proposed strategy may be extended for detecting various intracellular targets.Collectively,this strategy provides a valuable sensing platform for biomarkers analysis in living cells,thus having great potential for early clinical diagnosis and therapeutic evaluation.
文摘Dear Editor,CRISPR-Cas9 (clustered regularly interspaced short palin- dromic repeats-CRISPR associated) systems have been harnessed for kinds of genome manipulation, including gene editing, transcription regulation, and chromosome loci imaging (Dominguez et al., 2016; Komor et al., 2017). A typical engineered CRISPR-Cas9 system is composed of a Cas9 protein and a single guide RNA (sgRNA), which could form a protein/RNA complex to recognize and cleave DNA sequence (Hsu et al., 2014; Wright et al., 2016).