Semi-implantable device based on multiplexed microfilament electrode cluster for continuous monitoring of physiological ions

Modern medicine is increasingly interested in advanced sensors to detect and analyze biochemical indicators.Ion sensors based on potentiometric methods are a promising platform for monitoring physiological ions in biological subjects.Current semi-implantable devices are mainly based on single-parameter detection.Miniaturized semi-implantable electrodes for multiparameter sensing have more restrictions on the electrode size due to biocompatibility considerations,but reducing the electrode surface area could potentially limit electrode sensitivity.This study developed a semi-implantable device system comprising a multiplexed microfilament electrode cluster(MMEC)and a printed circuit board for real-time monitoring of intra-tissue K^(+),Ca^(2+),and Na^(+)concentrations.The electrode surface area was less important for the potentiometric sensing mechanism,suggesting the feasibility of using a tiny fiber-like electrode for potentiometric sensing.The MMEC device exhibited a broad linear response(K^(+):2–32 mmol/L;Ca^(2+):0.5–4 mmol/L;Na^(+):10–160 mmol/L),high sensitivity(about 20–45 mV/decade),temporal stability(>2weeks),and good selectivity(>80%)for the above ions.In vitro detection and in vivo subcutaneous and brain experiment results showed that the MMEC system exhibits good multi-ion monitoring performance in several complex environments.This work provides a platform for the continuous real-time monitoring of ion fluctuations in different situations and has implications for developing smart sensors to monitor human health.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Preparation of Silk-Like PET Microfilament and Its Properties

The polyester microfilament was prepared by means ofnormal fully drawn yarn(FDY)equipment.The effectof water content in chips,the molecular weight of thechips,the jet stretch ratio and the lateral blow on thespinnability and the finest titre of filament that could bereach were discussed.Under convenient conditions,thefiber with linear density of about 1.4 dtex could be ob-tained.By means of wide angle X-ray diffractometer(WAXD),differential scanning calorimeter(DSC)andInstrontester,the structure and properties of the mi-crofilament were discussed.

The Distribution and Morphology Alterations of Microfilaments and Microtubules in Mesophyll Cells and Root-Tip Cells of Wheat Seedlings under Enhanced Ultraviolet-B Radiation

The distribution and morphology alterations of microfilaments and microtubules in the mesophyll cells and root-tip cells of wheat seedlings, which had been radiated by enhanced ultraviolet-B (10.08 KJ·m-2·d-1), were examined through the confocal laser scanning microscope (Model FV1000, Olympus, Japan). Microtubule was labeled with an indirect immunofluorescence staining method, and microfilament was labeled with fluorescein isothiocyanate-phalloidin (FITC-Ph) as probes. The results indicated that microtubules in mesophyll cells, compared with the controls, would be depolymerized significantly, and dispersed randomly showing some spots or short rods in the cytoplasm, under the enhanced UV-B radiation condition. The microtubule bundles tended to be diffused, and the fluorescence intensity of that significantly decreased. The distribution pattern of microfilaments, which usually arranged parallelly in control cells, was broken up by enhanced UV-B radiation. We further investigated the distribution and morphology of microtubules in root-tip cells during every stage of cell division, and found that these aberrant phenomena of microtubules were often associated with abnormal cell division. Our findings suggested that the distribution, morphology and structure of cytoskeleton in mesophyll cells and root-tip cells of wheat seedlings would be affected by enhanced UV-B radiation, which might be related to abnormal cell division caused by enhanced UV-B radiation as an extracellular signal.

Distribution and localization of microfilament cytoskeleton is regulated by EBP50

Objective:To explore the influence of EBP50(ezrin-radixin-moesin-binding phospho-protein-50) on microfilament cytoskeleton content and distribution in cultured Hela cells, and to investigate the relationship between the changes in microfilament cytoskeleton localization and EBP50 after PDGF(platelet-derived growth factor) stimulation, and to further clarify the molecular mechanism by which EBP50 suppresses tumor cell proliferation and migration.Methods:pBK-CMV-HAEBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were transfected into Hela cells.G418 at 350 mg/L was used to screen for cell clones stably expressing EBP50.Western blot was carried out to detect EBP50 expression.Similarities and differences in microfilament cytoskeleton content and distribution in Hela cells transfected with pBK-CMV-HA-EBP50 wild type recombinant plasmid and pBK-CMV-HA empty vector were also treated with PDGF(10 ng/mL and 20 ng/mL, 37 ℃, 15 min) and stained by rhodamine-labeled phalloidin to observe the distribution of microfilament cytoskeleton in the two groups.EBP50 protein distribution in PDGF-stimulated Hela cells was detected by immunofluorescence.Results:Western blot results confirmed that the EBP50 cDNA fragment could express EBP50 in cultured Hela cell lines and that cell lines stably expressing EBP50 were successfully obtained.Western blot and fluorescence results showed that in the cell line transfected with empty vector, the microfilament cytoskeleton was thick, loose, multidirectional and displayed crossing arrangements.The content of microfilament cytoskeleton in the cell line transfected with pBK-CMV-HA-EBP50 was different from that found in the cell line transfected with empty vector.EBP50 expression enhanced microfilament cytoskeleton polymerization into compact thin filaments.Under the stimulation of PDGF, EBP50 migrated to the cell membrane from the cytosol together with microfilament cytoskeleton and co-localized there.Conclusion:EBP50 can change the distribution of microfilament cytoskeleton in cultured Hela cells and can also bind the microfilament cytoskeleton to the cell membrane under the stimulation of PDGF.EBP50 may play a role in the proliferation and migration of tumor cells by influencing the distribution and localization of microfilament cytoskeleton.

The alkaline tolerance in Arabidopsis requires stabilizing microfilament partially through inactivation of PKS5 kinase

High soil pH is harmful to plant growth and development. The organization and dynamics of microfilament （MF） cytoskeleton play important roles in the plant anti-alkaline process. In the previous study, we determined that alkaline stress induces a signal that triggers MF dynamicsdependent root growth. In this study we identified that PKS5 kinase involves in this regulatory process to facilitate the signal to reach the downstream target ME Under pH 8.3 treatment, the depolymerization of MF was faster in pks5-4 （PKS5 kinase constitutively activated） than that in wild-type plants. The inhibition of wild-type, pks5-1, and pks5-4 root growth by pH 8.3 was correlated to their MF depolymerization rate. When the plants were treated with phalloidin to stabilize MF, the high pH sensitive phenotype of pks5-4 can be partially rescued. When the plants were treated with a kinase inhibitor Staurosporine, the MF depolymerization rate in pks5-4 was similar as that in wild-type under pH 8.3 treatment and the sensitivity of root growth was also rescued. However, when the plants were treated with LaC13, a calcium channel blocker, the root growth sensitivity ofpks5-4 under pH 8.3 was rescued but MF depolymerization was even faster than that of plants without LaC13 treatment. These results suggest that the PKS5 involves in external high pH signal mediated MF depolymerization, and that may be independent of calcium signal.

Effects of Microfilamentous Reorganization on DG-PKC Pathway

Breakdown of phosphotidylinositol-4, 5-bisphosphate （PIP2） activates two distinct signal systems:production of inositol-1, 4, 5-trisphosphate （IP3） triggers release of calcium from intracellular stores, and release of diacylglycerol （DG） activates

The effects of microfilament and microtubule inhibitors and periodic electrical impulses on phloem transport in pea seedling

In phloem transport, whether protoplasmic activity participates in assisting sap flow in sieve element_companion cell complex has long been in debate. The present investigation assumed microfilament (MF) and microtubule (MT), the two constituents of the protoplasmic cytoskeleton, as motive force, and employed germinating pea seedling suspended in moist chamber as experimental material: the seed being the source; the elongating root, the sink. 14 C_labeled sucrose was added to the seed as indicator. The amount of sap transported from source to sink was measured by the increase in root elongation. The transport phloem was within the cylinder of the peeled root in the middle. The exposed cylinder was treated with MF inhibitor (cytochalasin B), or microtubule inhibitor (amiphos_methyl). Results showed that the sap influx into the elongating root, and the 14 C activity as well, was reduced by about one half in treatment with cytochalasin B, and much less by amiphos_methyl treatment. Similar effect was shown in electrical impulse treatment, which seems to disrupt the MF and MT configuration.

Stomatal Opening Induced by Acetylcholine Is Associated with Cytoskeletal Components

Acetylcholine (Ach) is a key component of animal cholinergic system. Recent experiments demonstrated that Ach, choline acetyltransferase, acetylcholinesterase and Ach receptors are present in all parts of plants and have many functions, including inducing stomatal movement. The authors' previous work has evidenced that microtubules and microfilaments are involved in regulating both stomatal closing and opening. The present investigation is to determine whether stomatal opening induced by Ach is associated with microtubules and microfilaments. The results showed that Ach could induce stomatal opening of Vicia faba L. with or without addition of KCl in the dark. Ach also stimulated protoplast swelling in a K +_free solution in the dark. However, the induction was partially suppressed when the strips and protoplasts were pretreated with either cytochalasin B, an inhibitor of F_actin polymerization, or oryzalin, an inhibitor of plant microtubule polymerization. Thus, our data suggest for the first time that stomatal opening induced by Ach is associated with the dynamics of microtubules and microfilaments.

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.

Actin Visualization in Living Immature 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 immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.

Integrin-linked kinase in gastric cancer cell attachment,invasion and tumor growth

AIM： To investigate the effects of integrin-linked kinase （ILK） on gastric cancer cells both in vitro and in vivo. METHODS： ILK small interfering RNA （siRNA） was transfected into human gastric cancer BGC-823 cells and ILK expression was monitored by real-time quan- titative polymerase chain reaction, Western blotting analysis and immunocytochemistry. Cell attachment, proliferation, invasion, microfilament dynamics and the secretion of vascular endothelial growth factor （VEGF） were also measured. Gastric cancer cells treated with ILK siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. RESULTS： Both ILK mRNA and protein levels were significantly down-regulated by ILK siRNA in human gastric cancer cells. This significantly inhibited cell attachment, proliferation and invasion. The knockdown of ILK also disturbed F-actin assembly and reduced VEGF secretion in conditioned medium by 40% （P 〈 0.05）. Four weeks after injection of ILK siRNA-transfected gastric cancer cells into nude mice, tumor volume and weight were significantly reduced compared with that of tumors induced by cells treated with non-silencing siRNA or by untreated cells （P 〈 0.05）. CONCLUSION： Targeting ILK with siRNA suppresses the growth of gastric cancer cells both in v/tro and /n vivo. ILK plays an important role in gastric cancer progression.

Application of VIGS system to explore the function of common wheat TaADF7

As an allohexaploid plant,common wheat has a complex gene structure,making it difficult to study its gene function.Virus-induced gene silencing(VIGS)is an important tool for the rapid analysis of plant gene function.In this study,the gene silencing system,namely,barley stripe mosaic virus(BSMV)-VIGS induced by BSMV was used to silence the wheat phytoene desaturase(TaPDS)and actin depolymerization factor(TaADF7)genes and determine the effect of gene silencing on wheat.TaPDS was used as an indicator gene to determine the feasibility of VIGS system,while TaADF7 was used as a test gene to determine its effect on wheat growth.Results showed that the leaves of tobacco and wheat were bleached by the mixture of pCaBS-α,pCaBS-β,and pCaBS-γ::TaPDS,indicating that the TaPDS gene was silenced,and the bleached leaves had physiological activity as determined by trypan blue staining.Therefore,the VIGS system was efficient and available.After the tobacco was treated with pCaBS-α,pCaBS-β,and pCaBS-γ::TaADF7,the viral suspension was obtained.The expression of TaADF7 gene was downregulated after wheat leaves were infected by friction,indicating that the expression of TaADF7 was silenced.Laser confocal scanning microscopy showed that the silencing of TaADF7 enhanced the fluorescence of microfilament skeleton in mesophyll protoplasts and significantly reduced the plant height.Results showed that TaADF7 affected cell division and plant growth by inhibiting microfilament depolymerization.In conclusion,the BSMV-VIGS system was used to silence wheat TaPDS and TaADF7 genes.Bleaching phenomenon was observed in wheat leaves after TaPDS silencing.After TaADF7 silencing,microfilaments in wheat mesophyll cells gathered into coarse bundles,which affected the dynamics of microfilaments and inhibited plant growth.

Dancing to a somewhat different rhythm: Cell migration along thenatural basement membrane

Much of our understanding of the events which underlie cell migration has been derived from studies of cells intissue culture. One of the components that mediates this process is the dynamic actin-based microfilament system that canreorganize itself into so-called stress fibers that are considered essential components for cell motility. In contrast, relativelyfew studies have investigated cell movement along an extracellular matrix (ECM) which is known to influence both cellularorganization and behavior. This opinion/viewpoint article briefly reviews cell migration during corneal endothelial woundrepair along the tissue’s natural basement membrane, Descemet’s membrane. Because the tissue exists as a cell monolayer itaffords one an opportunity to readily explore the effect of cell/matrix influences on cell motility. As such, cell movementalong this substrate differs somewhat from that found in vitro and migrating endothelial cells also demonstrate an abilityto move along the ECM without the benefit of having an organized actin cytoskeleton.

Role of Calcium in Glucagon Secretion

The role of calcium ions in the process by which nutrients affect glucagon secretion by pancreatic islet α-cells remains the matter of an apparently endless debate. In the prolongation of recent articles dealing with this matter, the present review draws attention to the dual role of Ca2+ as revealed by prior publications. In such a perspective, emphasis is placed on the increase in glucagon output in response to the omission of extracellular Ca2+ as recorded in the presence of D-glucose or 2-ketoisocaproate, the permissive role of extracellular Ca2+ in the positive secretory response to arginine or a mixture of fumarate, glutamate and pyruvate, and the effects of an organic calcium-antagonist on glucagon output. Considering the role currently ascribed to Ca2+ in the activation of motile events involved in stimulus-secretion coupling, attention is also given to the effects of cytochalasin B, D2O and mitotic-spindle inhibitors upon the secretory response of α-cells exposed to D-glucose in the absence or presence of arginine.

嗜热四膜虫接合生殖后期皮层细胞骨架蛋白的研究(英文)

用秋水仙素和细胞松弛素B处理接合期的嗜热四膜虫 ,以观察其对接合生殖期 ,尤其是接合后期的嗜热四膜虫皮层细胞骨架蛋白的影响。用秋水仙素处理的试验组的皮层细胞骨架蛋白组分中 34KD、37KD、46KD和 5 7KD蛋白的含量有明显改变 ,而用细胞松弛素B处理的试验组中 40KD和 74KD蛋白的含量改变较大。根据相关文献 ,作者推测 34KD、37KD、46KD、5 7KD蛋白是微管蛋白 ,而 40KD和 74KD可能是微丝蛋白。这些蛋白对嗜热四膜虫接合过程中的形态发生的重要作用有待进一步研究。

SCAB1 coordinates sequential Ca^(2+) and ABA signals during osmotic stress induced stomatal closure in Arabidopsis

Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability.Stomata are gateways of transpiration and gas exchange,the swift adjustment of stomatal aperture has a strong influence on plant drought resistance.Despite intensive investigations of stomatal closure during drought stress in past decades,little is known about how sequential signals are integrated during complete processes.Here,we discovered that the rapid Ca^(2+) signaling and subsequent abscisic acid(ABA)signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana,while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1(SCAB1)is the molecular switch for this entire process.During the early stage of osmotic shock responses,swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3(CPK3)and CPK6.The phosphorylation restrained the microfilament binding affinity of SCAB1,which bring about the Factin disassembly and stomatal closure initiation.As the osmotic stress signal continued,both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly.We further found that ABA signaling is indispensable for these attenuations,which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure.Notably,the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure.Taken together,our results support a model in which SCAB1 works as a molecular switch,and directs the microfilament rearrangement through integrating the sequentially generated Ca^(2+) and ABA signals during osmotic stress induced stomatal closure.

Nanomechanical vibration profiling of oocytes

The beginning of a mammalian life commences with a fertilized oocyte.The study of oocytes is certainly one of the most intriguing scientific questions of our time.Herein,we studied oocytes from a mechanical perspective and characterized the typical life activities of oocytes by nanomechanical vibrations.During the development of oocytes from the germinal vesicle(GV)stage to the zygotes,the GV stage oocytes induced a significant nanomechanical vibration,compared with the oocytes in meiosis I(MI)and meiosis II(MII)stages and zygotes.We analyzed the characteristics of mechanical vibrations of oocytes,including the amplitude as well as the frequency.It showed that the amplitude and frequency of nanomechanical vibrations induced by oocytes were caused by the cytoskeleton(microfilaments)and the distribution of metabolic characteristics(mitochondria)within oocytes.This work provides a new perspective for clinical quality assessment and basic research of oocytes,and can open new doors for development of life science.

Effects of nitrogen ion implantation on lily pollen germination and the distribution of the actin cytoskeleton during pollen germination

The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0±1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 1013 ions/cm2. Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.

A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium （rsa）. One of these mutants, rsa3-1, is hypersensitive to NaCI and LiCI but not to CsCI or to general osmotic stress. Reactive oxygen species （ROS） over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/ MUR3/KAMl-encoded xylogluscan galactosyltransferase regulates actin microfilament organization （and thereby con- tributes to endomembrane distribution） and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phal- Ioidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results sug- gest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.