Super-resolution structured illumination microscopy(SR-SIM)is an outstanding method for visualizing the subcellular dynamics in living cells.To date,by using elaborately designed systems and algorithms,SR-SIM can achi...Super-resolution structured illumination microscopy(SR-SIM)is an outstanding method for visualizing the subcellular dynamics in living cells.To date,by using elaborately designed systems and algorithms,SR-SIM can achieve rapid,optically sectioned,SR observation with hundreds to thousands of time points.However,real-time observation is still out of reach for most SIM setups as conventional algorithms for image reconstruction involve a heavy computing burden.To address this limitation,an accelerated reconstruction algorithm was developed by implementing a simplified workflow for SR-SIM,termed joint space and frequency reconstruction.This algorithm results in an 80-fold improvement in reconstruction speed relative to the widely used Wiener-SIM.Critically,the increased processing speed does not come at the expense of spatial resolution or sectioning capability,as demonstrated by live imaging of microtubule dynamics and mitochondrial tubulation.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
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
To determine the effects of microwave radiation at the molecular level as well as on the germination,growth and morphology of dry spores at the single-cell level.Dry Bacillus aryabhattai MCCC 1K02966 spores were micro...To determine the effects of microwave radiation at the molecular level as well as on the germination,growth and morphology of dry spores at the single-cell level.Dry Bacillus aryabhattai MCCC 1K02966 spores were microwave-treated at different powers and characterized using single-cell optical technology.As determined by laser tweezers Raman spectroscopy,the Ca^(2+)-dipicolinic acid content increased and nucleic acid denaturation occurred in response to microwave treatment.Livecell microscopy revealed that the germination and growth rates decreased as the microwave power increased.With respect to morphology,atomic force microscopy(AFM)demonstrated that spores became wrinkled and rough after microwave treatment.Furthermore,spores became smaller as the microwave power increased.Microwave treatment can damage DNA,and high-power microwaves can inhibit the germination of spores and reduce spore volumes.These results provide a new perspective on the responses of living single cells to microwave radiation and demonstrate the application of various new techniques for analyses of microorganisms at the single-cell level.展开更多
Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integ...Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.展开更多
Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors,ATP,and RNAs.In this regard,the stand-alone and genetic-code-independent peptide ligases constitute a new family of ren...Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors,ATP,and RNAs.In this regard,the stand-alone and genetic-code-independent peptide ligases constitute a new family of renegade peptide-bond makers.A prime example is butelase-1,an Asn/Asp(Asx)-specific ligase that structurally belongs to the asparaginyl endopeptidase family.Butelase-1 specifically recognizes a C-terminal Asx-containing tripeptide motif,Asn/Asp-Xaa-Yaa(Xaa and Yaa are any amino acids),to form a site-specific Asn-Xaa peptide bond either intramolecularly as cyclic proteins or intermolecularly as modified proteins.Our work in the past five years has validated that butelase-1 is a potent and versatile ligase.Here we review the advances in ligases,with a focus on butelase-1,and their applications in engineering bioactive peptides and precision protein modifications,antibody-drug conjugates,and live-cell labeling.展开更多
Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated v...Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.展开更多
Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission(AIE)characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nucl...Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission(AIE)characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nuclear permeability to live cells.This strategy has offered new opportunities for the development of probes with light-up ability and good signal-to-noise ratio.The selectivity or targeting specificity is determined by the peptide sequence,i.e.a nuclear localization signal that leads to nucleus imaging and a cell biomarker targeting peptide that offers specific light-up imaging of HT-29 cells.展开更多
In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusua...In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A （BFA）-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a re- markable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addi- tion, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.展开更多
基金supported by the National Natural Science Foundation of China (NSFC) (Nos. 62005208, 62135003, and 61905189)Innovation Capability Support Program of Shaanxi (No. 2021TD-57)+1 种基金China Postdoctoral Science Foundation (Nos. 2020M673365 and 2019M663656)National Institutes of Health Grant GM100156 to PRB
文摘Super-resolution structured illumination microscopy(SR-SIM)is an outstanding method for visualizing the subcellular dynamics in living cells.To date,by using elaborately designed systems and algorithms,SR-SIM can achieve rapid,optically sectioned,SR observation with hundreds to thousands of time points.However,real-time observation is still out of reach for most SIM setups as conventional algorithms for image reconstruction involve a heavy computing burden.To address this limitation,an accelerated reconstruction algorithm was developed by implementing a simplified workflow for SR-SIM,termed joint space and frequency reconstruction.This algorithm results in an 80-fold improvement in reconstruction speed relative to the widely used Wiener-SIM.Critically,the increased processing speed does not come at the expense of spatial resolution or sectioning capability,as demonstrated by live imaging of microtubule dynamics and mitochondrial tubulation.
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Lin He and Siyi Qiu received support from the National Natural Science Foundation of China(Grant No.91851210).
文摘To determine the effects of microwave radiation at the molecular level as well as on the germination,growth and morphology of dry spores at the single-cell level.Dry Bacillus aryabhattai MCCC 1K02966 spores were microwave-treated at different powers and characterized using single-cell optical technology.As determined by laser tweezers Raman spectroscopy,the Ca^(2+)-dipicolinic acid content increased and nucleic acid denaturation occurred in response to microwave treatment.Livecell microscopy revealed that the germination and growth rates decreased as the microwave power increased.With respect to morphology,atomic force microscopy(AFM)demonstrated that spores became wrinkled and rough after microwave treatment.Furthermore,spores became smaller as the microwave power increased.Microwave treatment can damage DNA,and high-power microwaves can inhibit the germination of spores and reduce spore volumes.These results provide a new perspective on the responses of living single cells to microwave radiation and demonstrate the application of various new techniques for analyses of microorganisms at the single-cell level.
基金supported by NIH-NEI grants to DRH(R01-EY018417,R01-EY024519)the Center for Zebrafish Research,University of Notre Dame,USA
文摘Sensing and responding to our environment requires functional neurons that act in concert. Neuronal cell loss resulting from degenerative diseases cannot be replaced in humans, causing a functional impairment to integrate and/or respond to sensory cues. In contrast, zebrafish（Danio rerio） possess an endogenous capacity to regenerate lost neurons. Here, we will focus on the processes that lead to neuronal regeneration in the zebrafish retina. Dying retinal neurons release a damage signal, tumor necrosis factor α, which induces the resident radial glia, the Müller glia, to reprogram and re-enter the cell cycle. The Müller glia divide asymmetrically to produce a Müller glia that exits the cell cycle and a neuronal progenitor cell. The arising neuronal progenitor cells undergo several rounds of cell divisions before they migrate to the site of damage to differentiate into the neuronal cell types that were lost. Molecular and immunohistochemical studies have predominantly provided insight into the mechanisms that regulate retinal regeneration. However, many processes during retinal regeneration are dynamic and require live-cell imaging to fully discern the underlying mechanisms. Recently, a multiphoton imaging approach of adult zebrafish retinal cultures was developed. We will discuss the use of live-cell imaging, the currently available tools and those that need to be developed to advance our knowledge on major open questions in the field of retinal regeneration.
基金supported by Academic Research Grant Tier 3(MOE2016-T3-1-003)from the Singapore Ministry of Education.
文摘Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors,ATP,and RNAs.In this regard,the stand-alone and genetic-code-independent peptide ligases constitute a new family of renegade peptide-bond makers.A prime example is butelase-1,an Asn/Asp(Asx)-specific ligase that structurally belongs to the asparaginyl endopeptidase family.Butelase-1 specifically recognizes a C-terminal Asx-containing tripeptide motif,Asn/Asp-Xaa-Yaa(Xaa and Yaa are any amino acids),to form a site-specific Asn-Xaa peptide bond either intramolecularly as cyclic proteins or intermolecularly as modified proteins.Our work in the past five years has validated that butelase-1 is a potent and versatile ligase.Here we review the advances in ligases,with a focus on butelase-1,and their applications in engineering bioactive peptides and precision protein modifications,antibody-drug conjugates,and live-cell labeling.
基金supported by the Hong Kong Health and Medical Research Fund grants COVID190121 to JF-WC and COVID190114 to D-YJthe Hong Kong Research Grants Council grants C7142-20GF and T11-709/21-N to D-YJ.
文摘Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.
文摘目的:对酵母发酵过程进行活菌电容值的在线监测,探索发酵过程中活菌细胞数量与代谢产物的关系。方法:在线监测A、B两组酵母细胞发酵过程中电容值与OD_(280)值数据。结果:发酵过程中,0~15 h电容值变化曲线呈对数增长,15~40 h电容值变化趋于平衡,0~40 h OD_(280)值呈持续增长趋势。结论:在培养体积固定的情况下,酵母细胞达到一定密度后,活细胞数量维持稳定,代谢产物持续增长。
基金the Singapore National Research Foundation(R279-000-444-281)the Singapore-MIT Alliance for Research and Technology(R279-000-378-592)the Economic Development Board(Singapore-Peking-Oxford Research Enterprise,COY-15EWI-RCFSA/N197-1)
文摘Fluorescent light-up probes comprising a tetraphenylethene unit with aggregation-induced emission(AIE)characteristics and a water-soluble peptide have been designed and synthesized which provide cell membrane and nuclear permeability to live cells.This strategy has offered new opportunities for the development of probes with light-up ability and good signal-to-noise ratio.The selectivity or targeting specificity is determined by the peptide sequence,i.e.a nuclear localization signal that leads to nucleus imaging and a cell biomarker targeting peptide that offers specific light-up imaging of HT-29 cells.
文摘In plant cells, the Golgi apparatus consists of numerous stacks that, in turn, are composed of several flattened cisternae with a clear cis-to-trans polarity. During normal functioning within living cells, this unusual organelle displays a wide range of dynamic behaviors such as whole stack motility, constant membrane flux through the cisternae, and Golgi enzyme recycling through the ER. In order to further investigate various aspects of Golgi stack dynamics and integrity, we co-expressed pairs of established Golgi markers in tobacco BY-2 cells to distinguish sub-compartments of the Golgi during monensin treatments, movement, and brefeldin A （BFA）-induced disassembly. A combination of cis and trans markers revealed that Golgi stacks remain intact as they move through the cytoplasm. The Golgi stack orientation during these movements showed a slight preference for the cis side moving ahead, but trans cisternae were also found at the leading edge. During BFA treatments, the different sub-compartments of about half of the observed stacks fused with the ER sequentially; however, no consistent order could be detected. In contrast, the ionophore monensin resulted in swelling of trans cisternae while medial and particularly cis cisternae were mostly unaffected. Our results thus demonstrate a re- markable equivalence of the different cisternae with respect to movement and BFA-induced fusion with the ER. In addi- tion, we propose that a combination of dual-label fluorescence microscopy and drug treatments can provide a simple alternative approach to the determination of protein localization to specific Golgi sub-compartments.
