Establishing delivery route-dependent safety and efficacy of living biodrug mesenchymal stem cells in heart failure patients

BACKGROUND Mesenchymal stem cells(MSCs)as living biopharmaceuticals with unique properties,i.e.,stemness,viability,phenotypes,paracrine activity,etc.,need to be administered such that they reach the target site,maintaining these properties unchanged and are retained at the injury site to participate in the repair process.Route of delivery(RoD)remains one of the critical determinants of safety and efficacy.This study elucidates the safety and effectiveness of different RoDs of MSC treatment in heart failure(HF)based on phase II randomized clinical trials(RCTs).We hypothesize that the RoD modulates the safety and efficacy of MSCbased therapy and determines the outcome of the intervention.AIM To investigate the effect of RoD of MSCs on safety and efficacy in HF patients.METHODS RCTs were retrieved from six databases.Safety endpoints included mortality and serious adverse events(SAEs),while efficacy outcomes encompassed changes in left ventricular ejection fraction(LVEF),6-minute walk distance(6MWD),and pro-B-type natriuretic peptide(pro-BNP).Subgroup analyses on RoD were performed for all study endpoints.RESULTS Twelve RCTs were included.Overall,MSC therapy demonstrated a significant decrease in mortality[relative risk(RR):0.55,95%confidence interval(95%CI):0.33-0.92,P=0.02]compared to control,while SAE outcomes showed no significant difference(RR:0.84,95%CI:0.66-1.05,P=0.11).RoD subgroup analysis revealed a significant difference in SAE among the transendocardial(TESI)injection subgroup(RR=0.71,95%CI:0.54-0.95,P=0.04).The pooled weighted mean difference(WMD)demonstrated an overall significant improvement of LVEF by 2.44%(WMD:2.44%,95%CI:0.80-4.29,P value≤0.001),with only intracoronary(IC)subgroup showing significant improvement(WMD:7.26%,95%CI:5.61-8.92,P≤0.001).Furthermore,the IC delivery route significantly improved 6MWD by 115 m(WMD=114.99 m,95%CI:91.48-138.50),respectively.In biochemical efficacy outcomes,only the IC subgroup showed a significant reduction in pro-BNP by-860.64 pg/mL(WMD:-860.64 pg/Ml,95%CI:-944.02 to-777.26,P=0.001).CONCLUSION Our study concluded that all delivery methods of MSC-based therapy are safe.Despite the overall benefits in efficacy,the TESI and IC routes provided better outcomes than other methods.Larger-scale trials are warranted before implementing MSC-based therapy in routine clinical practice.

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.

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.

甲醛固定对Live/Dead BacLight Bacterial Viability Kit死活细菌染液荧光显微计数海洋细菌的影响

利用Live/Dead BacLight Bacterial Viability Kit死活细菌染液对采自湛江东海大堤海水、沉积物细菌和大型海藻拟刚毛藻(Cladophoropsis zollingeri)内生细菌数量进行了甲醛固定处理前后的荧光显微计数对比分析。结果表明,新鲜样品(不加甲醛固定)、甲醛刚固定样品、甲醛固定1周样品和甲醛固定2周样品中海洋细菌数量差异不显著(p>0.05)。甲醛固定对Live/Dead BacLight Bacterial Viability Kit死活细菌染液荧光显微计数海洋细菌数量无显著影响,固定后的样品可在2周内完成计数。

F-Actin Visualization in Generative and Sperm Cells of Living Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.

Mechanical microenvironments of living cells: a critical frontier in mechanobiology

The fields of biomechanics and mechanobiology have long been predicated on the premise that mechanics governs cell behavior. However, over the past few years, a growing body of evidence has suggested that the mechanical environment very close to cells–the cell microenvironment–plays the most important role in determining what a cell feels and how it responds to tissue-level stimuli. To complicate matters further, cells can actively manipulate their microenvironments through pathways of recursive mechanobiological feedback. Harnessing this recursive behavior to understand and control cell physiology and pathophysiology is a critical frontier in the field of mechanobiology. Recent results suggest that the key to opening this scientific frontier to investigation and engineering application is understanding a different frontier: the physical frontier that cells face when probing their mechanical microenvironments.

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.

Fabrication of Silicon Carbide Quantum Dots via Chemical-Etching Approach and Fluorescent Imaging for Living Cells

A simple chemical-etching approach is used to prepare the silicon carbide quantum dots (QDs). The raw materials of silicon carbide (SiC) with homogeneous nanoparticles fabricated via self-propagating combustion synthesis are corroded in mixture etchants of nitric and hydrofluoric acid. After sonication and chromatography in the ultra-gravity field for the etched products, aqueous solution with QDs can be obtained. The microstructure evolution of raw particles and optical properties of QDs were measured. Different organophilic groups on the surface like carboxyl, oxygroup, and hyfroxy were produced in the process of etching. Fluorescent labeling and imaging for living cells of Aureobasidium pulluans were investigated. The results indicated that SiC QDs were not cytotoxic and could stably label due to the conjugation between organophilic groups of QDs and specific protein of cells, it can be utilized for fluorescent imaging and tracking cells with in vivo and long-term-distance. Moreover, mechanism and specificity of mark were also analyzed.

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-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.

Characteristics of Dynamic Load Response of a Fuel Cell with a Dead-ended Anode

The dynamic load characteristics of a proton exchange membrane fuel cell（PEMFC） with a dead-ended anode were studied. In a 70 h experiment, the effects of anode pressure, operating temperature, and relative humidity of the cathode on the performances of the fuel cell were investigated. The obtained results show that, with different relative humidity of the cathode at 65 ℃, dynamic loading has little effect on the performances of fuel cell and the electrochemically active surface area（ECSA）. However, the fuel cell operating under dynamic load is unstable when the relative humidity is 50%, and at 50 ℃ with 100% relative humidity, applying a dynamic load has a significant influence on the fuel cell performances. Scanning electron microscopy（SEM） showed that both the upstream and middle catalyst layers of the cell were unchanged, whereas the downstream cathode catalyst layer thinned as a response to dynamic load.

SmartFlare^(TM) is a reliable method for assessing mRNA expression in single neural stem cells

BACKGROUND One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells.On this matter,a novel platform called SmartFlare^(TM) has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts.Although fluorescence emission does not account for the spatial mRNA distribution,NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases,such as cancer,neurodegenerative pathologies or embryonic developmental disorders.AIM To investigate the potential of SmartFlare^(TM) in determining time-dependent mRNA expression of prominin 1(CD133)and octamer-binding transcription factor 4(OCT4)in single living cells through differentiation.METHODS Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres.For the in vitro differentiation,neurospheres were gently dissociated with Accutase solution.Single cells were resuspended in a basic medium enriched with fetal bovine serum,plated on poly-L-lysine-coated glass coverslips,and grown in a lapse of time from 1 to 4 wk.Live cell mRNA detection was performed using SmartFlare^(TM) probes(CD133,Oct4,Actin,and Scramble).All the samples were incubated at 37°C for 24 h.For nuclear staining,Hoechst 33342 was added.SmartFlare^(TM) CD133-and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach,taking into account the fluorescence intensity and the number of labeled cells.RESULTS In agreement with previous PCR experiments,a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro(DIV).Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern,also detectable in the contacting neural branches.From 15 to 30 DIV,only few cells showed a scattered fluorescent pattern,in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes.CONCLUSION SmartFlare^(TM) appears to be a reliable,easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model,preserving cell biological properties in anticipation of downstream experiments.

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.

Biosorption of Cd from Aqueous Solution by Live and Dead Biomass of Fusarium oxysporum Isolated from the Sludge of Abandoned Mine

Owing to exploring the biosorption mechanism of Fusarium oxysporum to Cd,the adsorption capacities of live and dead biomass of F.oxysporum strain KF2 were detected.The result showed both the live and dead biomass of strain KF2 could tolerate the Cd concentration up to 200 mg/L,and had the largest adsorption capacities for Cd,at 2.21 and 1.86 mg/g,respectively,with the inoculation amount of 3 g,the pH at 6,and the initial Cd concentration of 100 mg/L.The pseudo-second-order kinetic model(live biomass r^(2)>0.99,dead biomass r^(2)>0.90)was more suitable for describing the adsorption process of strain KF2.It indicated that the physicochemical adsorption on the cell surface might be the main pattern for Cd removal.Furthermore,the FTIR results showed that the main functional groups for cell wall to bind Cd were carboxyl,hydroxyl,amino,and phosphate.

Real-time observation of integrin bending/unbending conformational changes on living cells

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

Exact Traveling Wave Solutions for Nano-Solitons of Ionic Waves Propagation along Microtubules in Living Cells and Nano-Ionic Currents of MTs

In this work, the extended Jacobian elliptic function expansion method is used as the first time to evaluate the exact traveling wave solutions of nonlinear evolution equations. The validity and reliability of the method are tested by its applications to nano-solitons of ionic waves propagation along microtubules in living cells and nano-ionic currents of MTs which play an important role in biology.

3-82 Environmental Control of Live Cell Imaging System at Microbeam Facility

Live cell imaging has significantly changed our understanding to cell biology during the past 10 years. However, providing a suitable environment to keep cells heathy is still a challenge in live cell imaging experiments, and has great influence on the reliability of the experimental results. Many factors are needed to maintain cells healthy, such as temperature, pH, oxygen tension, CO2 and so on.

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.

3-79 Live Cell Imaging Study of XRCC1 Kinetics at the

DNA strand breaks can lead to cell carcinogenesis or cell death if not repaired rapidly and efficiently. An online live cell imaging system was established at the high energy microbeam facility at the Institute of Modern Physics (IMP) to study early and fast cellular response to DNA damage after high linear energy transfer (LET) ion radiation. HT1080 cells expressing XRCC1-RFP were irradiated with single high energy nickel ions, and time-lapse images of the irradiated cells were obtained online.