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 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.

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.

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.

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.

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.

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。

基于铁配合物的焦磷酸根离子荧光探针的构建及生物成像

以萘并[2,1-b]呋喃-2-碳酰肼和4-甲酰基-3-羟基-N-丁基-1,8-萘二甲酰亚胺为原料经缩合反应制备了一种新型配体ARC,配体ARC与Fe^(3+)按照1∶1络合制备了荧光探针ARC-Fe^(3+),通过高分辨率质谱(HR-MS)、核磁共振氢谱(1H NMR)对探针结构进行了表征。利用紫外-可见吸收光谱、荧光光谱等方法对ARC-Fe^(3+)的识别性能进行了研究。结果表明,探针ARC-Fe^(3+)可通过荧光“OFF-ON”高选择、高灵敏地识别焦磷酸根离子(PPi),探针ARC-Fe^(3+)识别PPi的检测限为1.12×10^(-8) mol/L,且可在60 s内完成对PPi的响应。探针ARC-Fe^(3+)已被成功应用于细胞和活鼠体内对PPi的荧光成像研究。

Application of Image Fusion Methods to Cell Imaging Processing

Aim To fuse the fluorescence image and transmission image of a cell into a single image containing more information than any of the individual image. Methods Image fusion technology was applied to biological cell imaging processing. It could match the images and improve the confidence and spatial resolution of the images. Using two algorithms, double thresholds algorithm and denoising algorithm based on wavelet transform,the fluorescence image and transmission image of a Cell were merged into a composite image. Results and Conclusion The position of fluorescence and the structure of cell can be displyed in the composite image. The signal-to-noise ratio of the exultant image is improved to a large extent. The algorithms are not only useful to investigate the fluorescence and transmission images, but also suitable to observing two or more fluoascent label proes in a single cell.

一种用于过氧亚硝酸根检测的近红外比率荧光探针的研制

设计合成了一种基于半花菁的近红外比率荧光探针分子Cy-P,并将其用于活细胞中的外源性和内源性过氧亚硝酸根(ONOO-)荧光成像检测.研究结果表明:探针分子Cy-P自身具有较大的π共轭结构,其最大发射波长在近红外区域;加入ONOO-后,强氧化性的ONOO-会导致Cy-P的π共轭体系被破坏,并生成蓝色荧光物质,最大发射波长蓝移了268 nm;探针分子Cy-P对ONOO-线性检测范围为0~15μmol/L,检测下限为13 nmol/L;该探针分子对ONOO-的识别性能优于其他各种可能干扰的生物分析物;该探针分子具有低细胞毒性,适用于活细胞中的外源性和内源性ONOO-的成像检测.

解析疫苗接种后抗体记忆的维持:进展与挑战

“首次感染可以为二次感染提供免疫保护”的生物学基本原理推动近代疫苗的诞生与使用。这种免疫保护的基础是抗体记忆,然而各种疫苗引起的有效抗体应答的持续时间各不相同。临床研究发现,接种减毒活疫苗后引起的中和抗体滴度通常水平较高且持续时间久。但减毒活疫苗存在难以回避的安全性风险,因此有必要进一步研究高水平、高质量抗体记忆的产生机制与调控因素,以开发兼具保护性与安全性的新型疫苗。抗体记忆的维持主要依赖于长寿浆细胞介导的抗体分泌,以及记忆B细胞在二次感染时介导的快速记忆性抗体应答。抗原结构与抗原效价、疫苗的抗原载量与免疫策略、病原体相关物质与疫苗佐剂等因素均可通过影响生发中心反应调控抗体记忆的持续时长。本文将梳理疫苗中各成分及因素对抗体记忆生成和维持的相关机制,并尝试讨论目前疫苗学和基础免疫学领域对此方面仍存有的问题与挑战。

基于图像识别的酵母活细胞率快速测定系统

酵母活细胞率作为评判活性干酵母质量优劣的重要指标之一,准确快速地检测出结果显得尤为重要。本文针对传统方法进行活细胞率检测存在效率低、过程复杂等问题开发了一款计数软件,并将血球计数板替换为96孔细胞培养板进行计数。对比人工计数和软件计数在检测酵母活细胞率的误差以及采用不同容器进行检验的效率,结果表明,相对于人工计数,软件计数耗时更短,且误差较小;采用96孔细胞培养板批量检测比采用血球计数板耗时更短。

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .

Iron-Imprinted Single-Atomic Site Catalyst-Based Nanoprobe for Detection of Hydrogen Peroxide in Living Cells

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.

Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

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.