Multiplexed stimulated emission depletion nanoscopy(mSTED)for 5-color live-cell long-term imaging of organelle interactome

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.

Recent advances in optical techniques for dynamically probing cellular mechanobiology

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.

Recent advances in living cell nucleic acid probes based on nanomaterials for early cancer diagnosis

The early diagnosis of cancer is vital for effective treatment and improved prognosis. Tumor biomarkers, which can be used for the early diagnosis, treatment, and prognostic evaluation of cancer, have emerged as a topic of intense research interest in recent years. Nucleic acid, as a type of tumor biomarker, contains vital genetic information, which is of great significance for the occurrence and development of cancer. Currently, living cell nucleic acid probes, which enable the in situ imaging and dynamic monitoring of nucleic acids, have become a rapidly developing field. This review focuses on living cell nucleic acid probes that can be used for the early diagnosis of tumors. We describe the fundamental design of the probe in terms of three units and focus on the roles of different nanomaterials in probe delivery.

Live cell imaging of genomic loci using dCas9-SunTag system and a bright fluorescent protein

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）.

Encapsulation of live cells by metal-organic frameworks for viability protection

In this study, a yeast@ZIF-8 core–shell composite material was successfully synthesized under room temperature in aqueous solution. The ZIF-8 shell endowed the inner yeast cells with a considerably extended lifetime without any nutrients at 4°C. Compared with the bare yeast cells, most coated yeast cells were kept alive even when cultured in zymolyase solution for 3h. Furthermore, the encapsulated yeast cells could be reactivated and regrown by dissolving the ZIF-8 shell with competitive coordination interactions.

AIEgen based light-up probes for live cell imaging

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.

3D Bioprinting with Live Cells

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.

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

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.

Synthesis of water-soluble dye-cored poly(amidoamine) dendrimers for long-term live cell imaging

Hydrophilic dendrimers, especially poly（amidoamine）（PAMAM） dendrimers are widely applied in modifying fluorescent dyes to endow them with water solubility and biocompatibility for biologic fluorescence imaging.Common preparation strategies of fluorescent dendrimers including encapsulating dyes or attaching dyes at periphery of dendrimers might cause uncertain constituent and lower biocompatibility. Here, we have developed a series of watersoluble fluorescent dendrimers with dye as core and fanshaped PAMAM as arms. Carboxylated perylene bisimides（PBI） dye and squarylium indocyanine（SICy） dye were conjugated with PAMAM dendrons by amidation to obtain a series of fluorescent dendrimers with enhanced water-solubility. Two PBI based dendrimers（PBI-G2.5 and PBI-G1.5）were chosen as model compounds for further optical, selfassembly and biological studies. In aqueous environment,PBI-G2.5 exhibited strong fluorescence, small size（~30 nm）and slightly positive surface charge（~2.46 mV）, which are ideal for biomedical applications. In vitro assays demonstrated that PBI-G2.5 nanoparticles accumulated in the cytoplasm of He La cells with rapid cellular uptake. The strong fluorescence in He La cells remained for over 48 h. To conclude, our study provides an effective strategy for preparing water-soluble fluorescent dendrimers towards long-term live cell imaging.

Live imaging of the effects of fucoidan on cell proliferation for laboratory instruction

The aim of this study is to introduce live cell imaging and its applications for the evaluation of the effects of fucoidan, a fucose-enriched sulfated polysaccharide, on the proliferation of cultured cells in vitro. In this study, long-term time- lapse observation （87 h） of the effects of fucoidan was conducted using BioStation CT, an integrated cell culture observation system. In contrast, the effects of heparin, which has a similar structure to fucoidan, were observed to distinguish the differences between the two chemicals. At the same time, the viability of the floating cells detached by fucoidan in the medium was measured by culturing them again in the absence of fucoidan. Finally, total internal reflection fluorescence microscopy （TIRF） was used to confirm when the detachment of the cells by fucoidan occurred. The results indicate that the inhibitory effects of fucoidan on the proliferation of cells are dose-dependent （from 0.125 mg/ml to 1.0 mg/ml）. Fucoidan also causes cell detachment without killing all the cells within 24 hours. The cell detachment did not occur until after half an hour, as observed under the TIRF microscope. Combined with our previous study, the findings suggest that the inhibition of calcium responses by fucoidan may be one of the mechanisms underlying its inhibition of cell proliferation, which is responsible for the death of cancer cells. Cell proliferation can be visualized in the real time and the images can provide important information regarding when and how the cells grow and proliferate.

Recent advances in small molecule fluorescent probes for simultaneous imaging of two bioactive molecules in live cells and in vivo

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.

Live-cell imaging:new avenues to investigate retinal regeneration

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.

Analysis of CD8^+CD28^-T-suppressor cells in living donor liver transplant recipients

BACKGROUND:Human CD8 + CD28 - T-suppressor(Ts) cells have been considered to indicate a reduced need for immunosuppression in pediatric liver-intestine transplant recipients and recipients of deceased heart-kidney transplants.However,in adult-to-adult living donor liver transplantation(A-A LDLT)little information is available and the clinical significance is still unknown. METHODS:Flow cytometry was used to detect the population of CD8+CD28 -Ts cells present in peripheral blood in A-A LDLT recipients(n=31),patients with end- stage liver disease(n=24)and healthy controls(n=19). Meanwhile,we tested the graft function and trough levels of immunosuppression in recipients.The clinical and follow- up data of 31 transplant recipients were analyzed. RESULTS:Compared with diseased controls(P=0.007) and healthy individuals(P=0.000),a notable expansion of CD8 + CD28 - Ts cells was found in recipients of A-A LDLT.This was associated with graft function,levels of immunosuppression and rejection episodes. CONCLUSIONS:To monitor the CD8 + CD28 - Ts cells levels is important to evaluate the immune state of recipients. Meanwhile,it is also important to promote expansion of CD8+CD28 -Ts cells in recipients of A-A LDLT,not only to sustain good graft function and decrease the dosage of immunosuppressants,but also to reduce the occurrence of rejection.

Target-activated and ratiometric photochromic probe for “double-check” detection of toxic thiols in live cells

Photochromic molecules can achieve reversible isomerization upon alternate light irradiations, which offers a great opportunity to improve the precision of analytes detection and imaging in complicated biological environments. Previous reported photochromic probe exhibited only mono-color switching and an initially fluorescence-ON state that may cause high background signal and impose an adverse impact on the desired sensing precision. To overcome this set-back, we developed a novel photochromic probe with an analyte-activation mode for ratiometric sensing of toxic thiols in both real water samples and live cells. The dynamic dual-fluorescence signal is released only after the fast and selective cleavage of the 2,4-dinitrophenyl sulfonate by the targeted thiophenol derivatives. Consequently, a "double-check" with synchronized dual-fluorescence blinking for analyte detection is successfully employed upon alternate light triggers with rapid response(k=7.2×10^(-2) s^(-1)), high sensitivity(LOD=6.1 nM) as well as selectivity of thiophenol derivatives over other common thiol species(e.g., GSH, Cys and Hcy). The photochromic probe was successfully introduced to the fast and on-site detection of highly toxic thiophenols in real waste water samples. Moreover, by using confocal laser-scanning microscopy(CLSM) and flow cytometric analysis, the potential applications of this ratiometric photochromic probe for trace toxic thiol sensing in live cells are examined.

REGULATION OF PRODUCTION OF PARATHYROID HORMONE-RELATED PEPTIDE(PTHrP)AND EXPRESSION OF ITS mRNA IN A HUMAN LIVER-DERIVED CELL LINE

Physiologic roles of PTHrP remain elusive,but some have implied a role of growth and differentiation.Since intestinal epithelial cell show orderly growth and differentiation as they proliferate in the crypt and migrate to the villus tip,we asked whether they might exhibit differences in expression of mRNA for either PTHrP or its receptor.AT/PCR was used to generate cDNA probe for either PTHrP or the PTH/PTHrP receptor.Total RNA was prepared from epithelial cells isolated form various region of rat gut and epithelial cell lines.derived from rat crypt(IEC-6)and human colon(LoVo)as wellas cell fractions taken sequentially along the villus-crypt axis of rat jejunum.The 1.6kb mRNA for PTHrP was detected in epithelia from all regions of rat gut(duodenum,jejunum,ileum,colon),in all fractions along the iejnnal villus tipcrypt axis,and in both cell lines.Likewise mRNA for the PTH/PTHrP receptor also was expressed,lbeit at lower level,in all regions,along the villus,and in both cell lines. Interestingly,while in kidney(positive control)two transcripts(1.5 & 2.4 kb)were detected as other reported,in intestinal epithelia and cell lines,only 1.skb transcript was evident.We conclude that mRNAs for both PTHrP and PTH/PTHrP receptor are expressed throughout the gut and that no obvious pattern of expre.ssion emerges from examining epithelia or cell lines representing different stage of differentiation. The role of PTHrP in gut epithelia remains to be defined.

Lived Experience of Sickle Cell Patients during and after Crisis

Aims and Objectives: To understand the lived experience and needs of patients with sickle cell disease during and two weeks after their crisis and identify the obstacles faced by patients while they are in the hospital. Background: Although there is no specific data of a number of affected individuals with sickle cell disease in Oman based on their age, the majority of the Omani population are youth. This category of the population is either in their high school or working in the governmental or private sector in the country. When the most productive category of the population are getting frequently absent due to sickle cell crisis and complication of sickle cell crisis from their work, this leads to huge financial and human resource burden. Design: Phenomenology. Method: This qualitative descriptive research was conducted using face-to-face interviews based on an interview protocol. The interview protocol was developed by the authors based on a framework called domains of well-being. Twenty adult patients have been recruited for the interview after meeting inclusion criteria and were asked about their well-being and lived experience during sickle cell crisis. Authors used SRQR checklist in reporting the study. Results: Thirteen themes were identified related to patients’ lived experience and their well-being during sickle cell crisis. Patients reported physical, emotional, social, and spiritual alteration. Major themes that emerged are communication, medical team interpretation of genuine pain, Emotional disturbance during the crisis, What does this study contribute to the wider global clinical community? Nurses and doctors should use therapeutic communication when dealing with sickle cell patients. Nurses should establish rapport and trust with patients. In each health care setting, there should be a social worker to deal with patients with chronic illness social relationships between the patient, family and friends, post-discharge status, spiritual and Islamic activities, and physical abilities. Conclusion: Participants’ physical and psychological statuses were mostly affected. Moreover, participants experienced extreme emotional disturbance during a painful crisis. However, it was not well understood why participants experienced post sickle cell crisis symptoms which need to be further investigated. Relevance to Clinical Practice: Understanding the lived experience of sickle cell patients may help improve nursing and medical care provided to them and enhance better outcomes for patients. These findings made the nurses and physicians plan a strategy of treating sickle cell patients using a holistic approach.

Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

基于智能化控制的高效活细胞成像技术

为提升活细胞功能研究的效率与准确性,显微成像平台研发了1款智能化控制精准加样系统。自主研发的加样系统具备远程自动化控制功能,能实现精准的药物定时定点添加。经研究和测试,该系统展现出卓越的通用性,能与常见的光学成像设备搭载使用,并能满足从短期到长期活细胞功能研究的各种实验要求。在活细胞连续成像的过程中,采用智能化与精准化的控制方法,可显著提升实验的准确性、稳定性和效率,尤其是能确保关键数据的完整性。该研究工作有效促进了细胞功能等关键科研领域的深入探索,成功增强了多台光学成像仪器的测试能力和应用价值,同时也推动了创新人才的成长和高标准科研平台的搭建。

在线活细胞定量检测模型的建立及其在辅酶Q10中的应用

在类球红细菌发酵生产辅酶Q10的过程中,其菌体生长代谢情况及产物合成与有活力的细胞量密切相关。活细胞传感仪通过利用细胞的介电特性,能够检测发酵液中的活细胞量,且检测结果不受死细胞、气泡的影响。在辅酶Q10的发酵过程中,运用活细胞传感仪采用全频扫描模式对检测到的电容值进行校正,校正后的电容值与CFU具有良好的线性关系,R^(2)=0.995,可以准确预测发酵液中菌体生长状态和活细胞量。基于电导率和校正后的电容值对辅酶Q10发酵过程中补料策略进行优化,辅酶Q10产量最终达到3420 mg/L。

罗伊氏乳杆活菌和热灭活菌对小鼠抑郁样行为的影响及作用机制

为了探究罗伊氏乳杆活菌与灭活菌对小鼠肠道菌群失衡引起的抑郁样行为的作用及作用机制,采用头孢曲松钠(Ceftriaxone sodium,CRO)处理构建肠道菌群失衡小鼠抑郁模型,并用罗伊氏乳杆活菌(Live Lactobacillus reuteri,Live LR)和热灭活菌(Heat-killed Lactobacillus reuteri,HK LR)进行干预。采用ELISA试剂盒测定5-羟色胺、5-羟基吲哚乙酸、色氨酸和皮质酮质量浓度;通过H&E染色观察结肠组织形态;通过高通量测序和生物信息学分析对肠道微生物组进行分析。结果表明:Live LR和HK LR干预缓解了小鼠抑郁样行为,修复了CRO引起的肠道菌群失调,并减少了结肠组织炎细胞浸润。两种益生菌的作用机制存在差异,Live LR侧重于肠道菌群的重塑,而HK LR则通过其代谢产物直接发挥抗炎作用。