Rate Of Fluorescence Recovery Research Articles

Effect of different crystal forms of MnO2 quenchers on the sensitivity of copper nanoclusters and their use in acidphosphatase activity.

Highly stable copper nanocluster (CuNCs) with aggregation-induced emission (AIE) properties was synthesized. α-, β-, and γ- MnO2 were utilized as quenchers, with CuNCs fluorescence quenching of 48.9%, 91.5%, and 96.6%, respectively. L-ascorbate-2-phosphate (AAP) was hydrolyzed by acid phosphatase (ACP), and ascorbic acid (AA) was formed. Then, MnO2 could be restored by AA, and the fluorescence of the CuNCs could be restored. An on-off-on detection platform with a high signal/noise ratio was constructed for the sensing of ACP. The fluorescence recovery rate of the CuNCs was related to the crystal forms of MnO2. Then, the equilibrium constants (K) for the reaction between AA and MnO2 were calculated to evaluate the reaction process. Compared with the K values of CuNCs/α-MnO2 and CuNCs/γ-MnO2, the K values for AA and β-MnO2 were maximum. The CuNCs/β-MnO2 system exhibited optimal fluorescence recovery for the sensitive detection of ACP. In the concentration range 0.005-0.06 U/mL, the detection limit was 0.0028 U/mL. The determination of serum ACP levels also revealed satisfactory results. This study provides novel insights into enhancing the sensitivity of the determination of quenchers in different crystal form.

EB1 Increases the Dynamics of Tau Droplets and Inhibits Tau Aggregation: Implications in Tauopathies.

EB1, a microtubule plus end-tracking protein (+TIP), regulates microtubule dynamics. Recent evidence indicates cross-talk between EB proteins and tau, a microtubule-associated neuronal protein that is important for the growth and stability of microtubules. We investigated the interaction between tau and EB1 and the effect of binding of EB1 on tau function and aggregation. EB1 colocalized with tau in SH-SY5Y cells and coimmunoprecipitated with tau. Further, purified EB1 impaired the ability of adult tau to induce tubulin polymerization in vitro. EB1 bound to tau with a dissociation constant of 2.5 ± 0.7 μM. EB1 reduced heparin-induced tau aggregation with a half-maximal inhibitory concentration of 4.3 ± 0.2 μM, and increased the dynamics of tau in phase-separated droplets. The fluorescence recovery rate in tau droplets increased from 0.02 ± 0.01 to 0.07 ± 0.03 s-1, while the half-time of recovery decreased from 44.5 ± 14 to 13.5 ± 6 s in the presence of 8 μM EB1, suggesting a delay in the transition of tau from the soluble to aggregated form in tau liquid-liquid phase separation. EB1 decreased the rate of aggregation and increased the critical concentration of tau aggregation. Dynamic light scattering, atomic force microscopy, dot blot assays, and SDS-PAGE analysis showed that EB1 inhibited the formation of oligomers and higher-order aggregates of tau. The data suggest a novel role for EB1 as a regulator of tau function and aggregation, and the findings indicated the role of the EB family proteins in neuronal function and neurodegeneration.

Photophysical Study of Heteroatom-Doped Carbon Dots-MnO2-Based Nanosensor: Selective Detection of Glutathione in the Nanomolar Level.

Heteroatom doping on carbon dots (Cdots) has been developed as an efficient approach to modify its optical and electronic properties. The four different types of heteroatom-doped Cdots (undoped Cdots (u-Cdots, nitrogen-doped Cdots (N-Cdots), sulfur-doped Cdots (Cdots), nitrogen, sulfur codoped Cdots (N, S-Cdots)) have been synthesized through a simple heat treatment of 5 min. Among four different heteroatoms doped nanosensors, N, S-Cdots with MnO2 nanospheres (Mn NS) showed one of the best fluorescents "on-off-on" nanosensors for selective sensing of glutathione (GSH) and cell imaging. N, S-Cdots showed a high fluorescence quantum yield, good photostability, ionic strength, and pH stability. N, S-Cdots with Mn NS demonstrated extremely high fluorescence quenching efficiency and the maximum fluorescence recovery rate after adding GSH to the produced solution. The photophysical study of N, S-Cdots-Mn NS used as a sensor confirms the inner filter effect (IFE) quenching mechanism between them. The developed sensor has an 80 nM limit of detection (LOD) for GSH. The heteroatom-doped framework of Cdots plays a significant role in the sensitive detection of GSH. N, S-Cdots-Mn NS have good permeability, biocompatibility, and low toxicity, due to which it was used in the intracellular imaging of GSH in living cells. The prepared sensor is rapid, economical, less toxic, and highly applicable in diagnosing diseases.

Smart flared-nanokites with ultra-high fluorescence enhancement for multiplexing virus DNA biosensing

A label-free sensing paradigm based on smart flared-nanokites is developed for multiplexed detection of virus DNA. A nanokite consists of silver nanoclusters beacons (Ag NCBs) immobilized on gold nanoparticle (Au NP) core. Ag NCB, possessing hairpin-shaped configuration, includes three functional domains: silver nanoclusters (Ag NCs) as the reporter, conjugated loop as recognizing element, and stem attached thiol. Via thiol-gold bond, Ag NCBs self-assemble on Au NP and form Ag NCBs/Au NP. In this conformation, close proximity of Ag NCs to Au NP leads to quenching fluorescence owing to energy transfer. However, upon recognizing loop binds to target, concomitant flying of Ag NCs from Au NP can be monitored as corresponding fluorescence enhancement, termed a flared-nanokite. Interestingly, ultra-high fluorescence recovery rate is calculated as 4379.2% (1300.4%) for G-/R-nanokite. Moreover, through by bicolor Ag NCBs simultaneously immobilize on Au NP, multicolor flared-nanokite for multichannel detecting DNAs has been successfully constructed based on multistimuli-responsive effect, demonstrating its promising application in accurate diagnosis and treatment.

A highly sensitive strategy for glypican-3 detection based on aptamer/gold carbon dots/magnetic graphene oxide nanosheets as fluorescent biosensor

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths in China. Glypican-3 (GPC3) is a specific antigen related to HCC, which is widely used in clinical detection as a reliable marker of HCC. In this paper, a highly sensitive homogeneous apatasensor was designed for GPC3 detection based on fluorescence resonance energy transfer (FRET) where the GPC3 aptamer labelled gold carbon dots (AuCDs-GPC3Apt) are used as a donor and magnetic graphene oxide (Fe3O4/GO) nanosheets are used as an acceptor. A one-step hydrothermal method was used to synthesize AuCDs to provide sufficient fluorescence. The FRET phenomenon exists between AuCDs-GPC3Apt and Fe3O4/GO, which weakens the fluorescence intensity of the whole system. When the target GPC3 is added to the FRET system, the fluorescent AuCDs-GPC3Apt binds to the GPC3 and forms a folded structure, which leads to AuCDs-GPC3Apt separation from Fe3O4/GO nanosheets. The Fe3O4/GO is then magnetically separated so that the fluorescence of free labelled AuCDs-GPC3Apt is restored. Under the optimum conditions, the fluorescence recovery rate is linearly correlated with the concentration of GPC3 (5-100ng·mL-1) and the detection limit is 3.01ng·mL-1 (S/N = 3). This strategy shows recoveries from 98.76 to 101.29% in real human serum samples and provides an immediate and effective detection method for the quantification of GPC3 with great potential applications for early diagnosis of HCC. A sensitive homogeneous FRET-based apatasensor was designed for GPC3 detection where the AuCDs-GPC3Apt is a donor and Fe3O4/GO nanosheets are an acceptor. The GPC3 fluorescent aptasensor combines wider output range with low cost, high specificity, and good anti-interference.

A fluorescence aptasensor based on GSH@GQDs and RGO for the detection of Glypican-3

Glypican-3 (GPC3), a heparin sulfate proteoglycan, is a potential diagnostic and therapeutic target for hepatocellular carcinoma. In this paper, a novel fluorescent aptasensor for GPC3 detection is constructed via glutathione@graphene quantum dots–labeled GPC3 aptamer (GSH@GQDs-GPC3Apt) as a fluorescence probe. First, GSH@GQDs is screened out with higher fluorescence intensity, which emits bright blue fluorescence under ultraviolet light. Then, the fluorescence-labeled GSH@GQDs-GPC3Apt probe is formed by the combination of amination GPC3Apt and GSH@GQDs using EDC/NHS coupled reaction. Under hydrogen bond and π-π interaction/stacking, the fluorescence of GSH@GQDs-GPC3Apt could be quenched by reductive graphene oxide (RGO) with the help of the photoinduced electron transfer and the fluorescence resonance energy transfer mechanism. In the presence of GPC3, the GSH@GQDs-GPC3Apt specifically recognizes and binds to GPC3, giving rise to the change of secondary structure of GPC3Apt to form the GPC3/GPC3Apt-GSH@GQDs complex, which would lead to the disintegration of the GSH@GQDs-GPC3Apt-RGO compound. Therefore, the energy transfer process is blocked and the fluorescence intensity is restored, enabling a highly sensitive response to GPC3. When the concentration of GPC3 is from 5.0 ng/mL to 150.0 ng/mL, the fluorescence recovery rate is well linearly related to GPC3 concentration with the limit of detection of 2.395 ng/mL (S/N = 3). This strategy shows recoveries from 98.31% to 101.89% in human serum samples and provides simple, fast and cheap analysis of GPC3, which suggests that it has great potential applications in clinical diagnosis for hepatocellular carcinoma.

Target-Activating and Toehold Displacement Ag NCs/GO Biosensor-Mediating Signal Shift and Enhancement for Simultaneous Multiple Detection.

Herein, we demonstrate that a new multicolor silver nanoclusters/graphene oxide (Ag NCs/GO) hybrid material, upon target response, undergoes a configuration transformation, based on entropy-driven enzyme-free toehold-mediated strand displacement reaction, achieving emission shift and enhancement. To realize the aim above, two different synthesis routes (route I and II) of synthesizing fluorescent Ag NCs for constructing toehold displacement Ag NCs/GO biosensor is designed and performed. Influenza A virus subtype genes (H1N1 and H5N1) as a model can efficiently initiate the operation of entropy-driven displacement reaction, resulting in activatable fluorescence. Red-emitting and green-emitting Ag NCs tethering the complementary sequence of H1N1 (pDNA1) and H5N1 (pDNA2) are indirectly immobilized on GO surface through binding with capture DNA (cDNA1 and cDNA2), respectively, forming multicolor pDNA-Ag NCs/GO nanohybrid materials. However, they do not exhibit nearly fluorescence signals attributed to energy transfer from donor Ag NCs to acceptor GO. Upon adding targets H1N1 and H5N1 (tDNA1 and tDNA2), pDNA1-Ag NCs and pDNA2-Ag NCs detach from GO, based on toehold-mediated strand displacement reaction, which interferes the energy transfer and leads to significant fluorescence enhancement. More interestingly, the activatable process is accompanied by remarkable hypsochromic shift (19 nm) or bathochromic shift (21 nm) emission with quite high fluorescence recovery rates (823.35% and 693.62%). Therefore, based on these phenomena, a novel multiple approach has been developed with the assistance of toehold displacement and Ag NCs/GO nanohybrid materials. As for the remarkable emission recovery and multichannel signal, the proposed approach displays the promising application prospect in accurate diagnosis and treatment.

A Rational design of multi-functional nanoplatform: Fluorescent-based "off-on" theranostic gold nanoparticles modified with D-α-Tocopherol succinate.

It is crucial to develop nanocarrier systems to detect and treat drug-resistant micro tumors to prevent recurrence and/or metastasis of cancer. Due to their exceptional features such as biocompatibility, easy surface modification, serving as imaging and therapeutic agent, gold nanoparticles (AuNPs) draw attention as theranostic agents. It is beneficial to combine AuNPs with a second imaging and/or treatment modality such as photodynamic therapy (PDT). PDT is a non-mutagenic treatment approach in which photosensitizer is activated with light, generating reactive oxygen species and/or free radicals to destroy tumor cells. With the aim of developing "off-on" theranostic system, citrate stabilized spherical 13nm AuNPs were densely coated with polyethylene glycol (PEG). To advance the theranostic feature of PEGylated AuNPs, they were further functionalized with FDA-Approved photosensitizer, Verteporfin (BPD-MA). Due to static quenching between BPD-MA and AuNPs as well as in between nearby BPD-MA molecules, the fluorescence of the ground state complex is quenched and the system is in "off" state. When BPD-MA molecules are cleaved from the AuNPs surface and diffuse away, fluorescence is recovered. Consequently, the system switches to the "on" state. Among the various mole ratios of BPD-MA carrying conjugates prepared, the most promising candidate was selected based on stability, quenching factor, and fluorescence recovery rate. The conjugate was further decorated with D-α-Tocopherol succinate (VitES) to increase the therapeutic efficacy of the theranostic agent via enhancing cellular uptake. Our results showed that it was possible to achieve as high as 80 times fluorescence quenching when the system was "off". As the system switched from "off" to "on" state, 51% of the fluorescence was recovered. When BPD-MA was immobilized on the PEGylated AuNPs, the phototoxic effect of BPD-MA increased twice against the MCF-7 cell line. Moreover, the developed system showed four times more phototoxicity than BPD-MA alone after it was decorated with VitES. Since the developed system is capable of dual imaging (computed tomography and fluorescence) and dual treatment (PDT and hyperthermia), it potentially offers superior imaging and therapy options for various types of in vitro/in vivo applications.

Abstract P-24: Microscopic Analyses of Liquid-Liquid Phase Separation Induced by Linker Histone H1.0

Background: Liquid-liquid phase separation (LLPS) that leads to the formation of temporary functional domains in cells plays an important role in the processes of chromatin condensation and gene regulation. Earlier, it was demonstrated that histone H1.4 can form LLPS droplets with DNA. In the present work, LLPS was studied for histone H1.0, which is mainly expressed in differentiated and non-dividing cells. H1.0 is involved in cancer development: its amount decreases with the progression of tumor cells to malignancy. Methods: LSM710 confocal microscope (Zeiss) equipped with the 40x/1.2W objective was used to image mixtures of H1.0 with Cy3/Cy5 labeled DNA or nucleosomes in fluorescent and transmitted-light channels at the excitation of 514 nm. The formation of condensates as a result of LLPS was confirmed by salt-jump and FRAP/FLIP experiments. Results: Condensates were not observed when the ratio of negative to positive charges (N/P) in the samples was >1. At N/P~0.7, optically homogeneous droplet-like condensates were found. The appearance of condensates, their size and shape depended on concentrations of H1.0 and DNA. LLPS condensates but not aggregates disappeared by salt-jump to 650 mM NaCl. FRAP/FLIP experiments revealed a moderate rate of fluorescence recovery (τ½22s) indicating moderate DNA mobility of the H1.0-mediated condensates. The appearance of condensates was also observed in the mixtures of H1.0, DNA and Cy3/Cy5-labeled nucleosomes. Nucleosomes were involved in the condensate formation and found to be 2-fold more mobile (τ½10 s) than DNA. Conclusion: LLPS-related properties of H1.0 were studied for DNA and nucleosomes in vitro. Comparison with H1.4 shows that H1.0 forms liquid condensates of approximately the same size. Our result also may indicate that chromatin retains pronounced dynamic properties in H1.0-induced droplets despite the fact that H1.0 induces the formation of more compact chromatin.

The preparation of supported lipid bilayers and its functional study

Supported lipid bilayers (SLBs) have been widely used in biomedical and bioengineering research in vitrobecause its structure and function are similar to natural cell membrane. A fluorescence recovery after photobleaching (FRAP) technique was used to measure the lateral diffusion of the SLBs composed of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3-［(N-(5-amino-1-carboxyp-entyl) iminodiacetic acid)］ (DGS-NTA) on the glass slide, and the effects of the DOPC-to-DGS-NTA ratio, small unilamellar vesicles (SUV) producing method, sizes of bleaching areas and concentrations of loading proteins on the SLBs fluidity and diffusion coefficient were studied systematically in this paper. The results demonstrated that: (1) SUV made by probe sonication exhibited more uniform and smaller size compared with that made by film extrusion, but the whole process of SLBs formation must not be exposed to air. (2) The fluorescence recovery rate and diffusion coefficient of the SLBs decreased with the increasing bleaching area size. With the mole ratio of DOPC to DGS-NTA decreasing from 98∶2 to 84∶16, the fluidity and fluorescence recovery degree decreased gradually, and the SLBs would lose its fluidity if the ratio reached to 82∶18. (3) The average fluorescence intensity of SLBs increased linearly with the loading protein concentration (10-40 nmol·L -1), and the protein showed good mobility on the SLBs. The study would provide a good platform of bio-membrane for further research on interactions among cell membrane molecules and subsequent signals response.

A fluorescence turn-on biosensor based on gold nanoclusters and aptamer for alpha fetoprotein detection

Alpha-fetoprotein (AFP), an important tumor marker, is widely used for the diagnosis of patients with germ cell tumors and hepatocellular carcinoma. Herein, a highly sensitive aptasensor for AFP detection was constructed based on fluorescence resonance energy transfer (FRET) between 5-carboxyfluorescein (FAM) and gold nanoclusters (AuNCs). AuNCs can effectively quench the fluorescence of FAM-AFP aptamer via strong coordination interactions. When AFP was introduced into the FAM-AFP aptamer-AuNCs FRET system, the AFP aptamer preferentially combined with AFP accompanied by conformational change, which greatly weakened the coordination interaction between the AFP aptamer and AuNCs. Thus, fluorescence recovery of FAM was observed. In the range of 10.0 -100.0 ng/mL, a linear relationship between the fluorescence recovery rate and the concentration of AFP was obtained with the detection limit of 6.631ng/mL. The highly sensitive FRET aptasensor with simple configuration showed promising prospect in detecting a variety of biomarkes.

Effects of bisphenol A on gap junctions in HaCaT cells as mediated by the estrogen receptor pathway.

Bisphenol A (BPA) is widely used as the raw material for the production of plastics and paper products. People can be exposed to BPA through dermal contact, particularly for cashiers in contact with thermal paper every day. BPA is a known endocrine disruptor that has been shown to be carcinogenic. Many tumors show weak gap junctional intercellular communication (GJIC) function. The aim of this study was to investigate the effects and possible mechanisms of BPA's action on GJIC of human HaCaT skin cells. The results showed that BPA increased cell proliferation rates, prolonged GJIC photobleaching fluorescence recovery times and reduced overall fluorescence recovery rates at 0.01, 0.1 and 1μm. BPA downregulated connexin (Cx)26 mRNA level at 0.1μm. Estrogen receptor (ER) antagonist ICI 182780 at 5nm partially blocked the above effects of BPA indicating involvement of the ER pathway with BPA exposure. However, BPA did not influence Cx43 mRNA and protein levels. Our immunofluorescence data showed that Cx26 was expressed in the cytoplasm and plasma membrane, and was involved in the formation of gap junctions between adjacent cells, while Cx43 was expressed only in the cytoplasm. Therefore, our data indicate that Cx26 gap junctions may be involved in the GJIC inhibition caused by BPA. In conclusion, our results indicate that BPA can promote human skin cell proliferation, inhibit skin cell GJIC function but not formation and downregulate Cx26 mRNA levels partially through the ER pathway. We hypothesize that BPA can exhibit carcinogenicity by inhibiting GJIC.

Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and draws great concern in health and food safety. A DNA aptamer against AFB1 having a stem-loop structure shows high binding affinity to AFB1 and promise in assay development for AFB1 detection. Based on the structure-switching property of theaptamer, we report an aptamer fluorescence assay for AFB1 detection. Aptamer with fluorescein (FAM) label at 5' end was used as affinity ligand, while its short complementary DNA (cDNA) with BHQ1 (black hole quencher 1) label at 3' end was used as a quencher. In the absence of AFB1, FAM-labeled aptamer hybridized with BHQ1-labeled cDNA, forming a duplex of cDNA and aptamer, resulting in fluorescence quenching of FAM. When AFB1 bound with aptamer, the BHQ1-labeled cDNA was displaced from aptamer, causing fluorescence restoration of FAM. We tested a series of FAM-labeled aptamers and BHQ1-labeled cDNAs with different lengths. The lengths of the aptamer stem and the cDNA, Mg2+ in binding buffer, and temperature had significant influence on the performance of the assay. Under optimized conditions, we achieved sensitive detection of AFB1 by using a 29-mer FAM-labeled aptamer and a 14-mer BHQ1-labeled cDNA, and the detection limit of AFB1 reached 0.2nM. The maximum fluorescence recovery rate of FAM-labeled aptamer caused by AFB1 was about 69-fold. This method enabled the detection of AFB1 in complex sample matrix, e.g., diluted wine samples and maize flour samples. This aptamer-based fluorescent assay for AFB1 determination shows potential for broad applications. Graphical abstract ᅟ.

Fluorescence photobleaching of microplastics: A cautionary tale

Fluorescence microscopy is an important step in visual identification of microplastics and is used to highlight white and transparent plastics that are otherwise easily missed or misidentified. Investigators using fluorescence must proceed with caution, however, as fluorescence photobleaching can significantly reduce the fluorescence output of samples within experimentally relevant time frames. We report on the photobleaching rate and subsequent lack of fluorescence recovery of five common plastics. Our results reveal statistically different photobleaching rates across plastic types. In the best-case scenario of low illumination intensity and a robust plastic, initial fluorescence intensity decayed by 10% in just 11(3) s and by 33% in 230(40) s. In all cases, fluorescence failed to recover more than 13(8)% in 3 h. These results indicate that significant bleaching can occur while searching a sample for plastics to identify and that the lack of recovery can compromise samples for further study.

Effect of paeoniflorin and menthol on membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity during transport of puerarin in Calu-3 cell

The aim of this research is to investigate the effects of paeoniflorin and menthol on the physiological function of Calu-3 cell membrane during the transport of puerarin. Calu-3 cell was used as the in vitro cell model to simulate nasal mucosa tissues, and the cell membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity were detected by fluorescence recovery after photobleaching(FRAP) and ultramicro enzyme activity testing, in order to explore the mechanism of compatible drugs on promoting puerarin transport. The results showed that when puerarin associated with low, middle and high concentration of menthol or both paeoniflorin and menthol, the fluorescence recovery rate was increased significantly, while Na⁺-K⁺-ATPase activity had no significant change and Ca²⁺-ATPase activity was enhanced significantly as compared with puerarin alone. Therefore, it was concluded that menthol had the abilit of promoting the transport and the mechanism might be related to increasing membrane fluidity and activating Ca²⁺-ATPase.

In Vivo Measurement of Glycine Receptor Turnover and Synaptic Size Reveals Differences between Functional Classes of Motoneurons in Zebrafish

The interplay between binding and unbinding of synaptic receptor proteins at synapses plays an important role in determining receptor concentration andsynaptic strength, with known links between changes in binding kinetics and synaptic plasticity. The regulation of such kinetics may subserve the specific functional requirements of neurons in intact circuits. However, the majority of studies of synaptic turnover kinetics have been performed in cultured neurons outside the context of normal circuits, and synaptic receptor turnover has not been measured at individual synaptic sites invivo. We quantified the distribution of glycinergic receptor dynamics using fluorescence recovery after photoconversion of synapses in intact zebrafish and correlated recovery kinetics to synaptic volume in two functionally distinct classes of cells: primary and secondary motoneurons. The rate of fluorescence recovery after photoconversion decreased with synaptic volume in both types of motoneurons, with larger synapses having slower recovery. Primary motoneurons had both larger synapses and associated slower recovery times than secondary motoneurons. Our results suggest that synaptic kinetics are regulated in concert with synaptic sizes and reflect the functional role played by neurons within their circuit.

Anisotropic Diffusion of Proteins in the Sarcoplasmic Reticulum of Skeletal Muscle

The way proteins move inside crowded organelles is determined by both the connectivity of the organellar lumen and the mutual interactions between molecules. To probe diffusional movement inside the SR we used FRAP of three proteins expressed in mouse paw muscles: the biosensor D1ER and two fluorescently tagged forms of calsequestrin (Casq1). Longitudinal diffusion (parallel to the fiber axis) was evaluated by the rate of fluorescence recovery in a rectangular bleached region, 10 μm wide and long enough to span the fiber width (∼40 μm). Diffusion in the transversal direction was evaluated with a bleached region of the same dimensions, placed longitudinally, right at the myofiber axis. The FRAP rates measured thus for D4cpV-Casq1 in resting myofibers corresponded (by theory and simulations) to an effective longitudinal diffusion coefficient DL of 0,012 μm2/s, while the transversal coefficient DT was 0,026 μm2/s. The difference was statistically significant. Faster transversal diffusion was also found for EYFP-Casq1. Additionally, the measurements evinced the presence of a large fraction of immobile Casq1, presumably polymerized within terminal cisternae. As reported elsewhere, the immobile fraction of Casq1 decreased, while the diffusional mobility of Casq1 and D1ER increased upon Ca2+ depletion in the SR. Electron microscopic images of resting myofibers demonstrate a greater connectivity of SR cavities in the transversal direction (i.e. along terminal cisternae) than longitudinally, to an extent consistent with and probably explaining in full the observed diffusional anisotropy. Regardless of the SR Ca2+ content, the diffusion coefficient values found for these proteins are much lower than the lowest values reported for proteins inside the ER, which probably reflects the intricacy of the SR structure and the polymeric state of calsequestrin in resting skeletal muscle. Supported by NIH grants AR049184 and GM111254.

Structural and functional changes in gap junctional intercellular communication in a rat model of overactive bladder syndrome induced by partial bladder outlet obstruction.

The aim of the present study was to investigate the association between connexin (Cx)43 levels and alterations in gap junctional mediation of intercellular communication in overactive bladder syndrome (OAB), and to examine the effects of connexin inhibitor on this condition. Adult female Wistar rats with OAB following partial bladder outlet obstruction (PBBO) (OAB group, n=37) and sham-operated rats (control group, n=17) were studied. The ultrastructure of the rat detrusor was observed by transmission electron microscopy and the protein expression levels of Cx43 were analyzed using western blot analysis. Furthermore, bladder detrusor cells in both groups were cultured and cells in the OAB group were randomly divided into ten groups. In nine of these groups, 18-β glycyrrhetinic acid (18β-GA) was administered at various doses and durations. All groups were compared using fluorescence redistribution after photobleaching and a laser scanning confocal microscope. Cystometry demonstrated that gap junctions were an abundant mechanism among adjacent cells, and Cx43 protein expression levels were increased in the OAB group following 6 weeks of obstruction, as compared with the control group. Mean fluorescence recovery rates in the OAB group were significantly increased, as compared with the control group (P<0.01). Mean fluorescence recovery rates were noted following 18β-GA administration. These results suggested that upregulation of Cx43 induces structural and functional alterations in gap junctional intercellular communication following PBOO, and connexin inhibitors may be a novel therapeutic strategy for the clinical treatment of OAB.

All-trans retinoic acid arrests cell cycle in leukemic bone marrow stromal cells by increasing intercellular communication through connexin 43-mediated gap junction

BackgroundGap junctional intercellular communication (GJIC) is typically decreased in malignant tumors. Gap junction is not presented between hematopoietic cells but occurred in bone marrow stromal cells (BMSCs). Connexin 43 (Cx43) is the major gap junction (GJ) protein; our previous study revealed that Cx43 expression and GJIC were decreased in acute leukemic BMSCs. All-trans retinoic acid (ATRA) increases GJIC in a variety of cancer cells and has been used to treat acute promyelocytic leukemia, but the effects of ATRA on leukemic BMSCs is unknown. In this study, we evaluated the potential effects of ATRA on cell cycle, proliferation, and apoptosis of leukemic BMSCs. Effects of ATRA on Cx43 expression and GJIC were also examined.MethodsHuman BMSCs obtained from 25 patients with primary acute leukemia, and 10 normal healthy donors were cultured. Effects of ATRA on cell cycle, cell proliferation, and apoptosis were examined with or without co-treatment with amphotericin-B. Cx43 expression was examined at both the mRNA and protein expression levels. GJIC was examined by using a dye transfer assay and measuring the rate of fluorescence recovery after photobleaching (FRAP).ResultsATRA arrested the cell cycle progression, inhibited cell growth, and increased apoptosis in leukemic BMSCs. Both Cx43 expression and GJIC function were increased by ATRA treatment. Most of the observed effects mediated by ATRA were abolished by amphotericin-B pretreatment.ConclusionsATRA arrests cell cycle progression in leukemic BMSCs, likely due to upregulating Cx43 expression and enhancing GJIC function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0212-7) contains supplementary material, which is available to authorized users.

Mechanism of regulating the resistance of prostate cancer PC-3 cells to cisplatin through enhancing cell gap junctions by evodiamine

Objective To evaluate the effect of the combination of cisplatin and evodiamine on the growth of PC-3 cells and gap junction intercellular communication(GJIC). Methods Human prostate cancer PC-3 cells in logarithmic growth phase were cultured in vitro and then treated with various concentrations of cisplatin(5,10,15,20μmol/L)or evodiamine(0. 4μmol/L)combined with various concentrations of cisplatin(5, 10, 15, 20μmol/L).The effects of the proliferation was investigated by cell counting kit-8(CCK-8)assay.Apoptosis rate, and cell cycle distribution were examined by Annexin V-fluoresceine isothiocyanate(FITC)/propidium iodide(PI) double staining flow cytometry.The expression of connexin43(Cx43) protein was detected by Western blotting.The changes in the GJIC function were observed by FRAP. Results Fluorescence recovery rate was(41. 93± 1. 30)%,(36. 32±2. 33)%,(30. 81±1. 43)%, and(22. 54±1. 70)% in cisplatin groups, and (69. 86±2. 16)%,(50. 03±5. 00)%,(45. 38±2. 59)%, and(34. 01±4. 01)% in cisplatin+ evodiamine groups with statistically significant differences within groups respectively(P< 0. 05 for all). Fluorescence recovery rate, apoptosis rate, and the expression of Cx43 protein in cisplatin+ evodiamine groups were significantly increased as compared with cisplatin groups(P< 0. 05 for all). Conclusion Evodiamine could recover the gap junction intercellular communication through increasing the expression of Cx43 protein and enhancing the cytotoxicity of cisplatin to prostate cancer PC-3 cells. Key words: Evodiamine; Cisplatin; Prostate cancer; Gap junctioninter cellular communication