Leiting Pan

Ultrasensitive flow sensing of a single cell using graphene-based optical sensors.

On the basis of the polarization-dependent absorption of graphene under total internal reflection, we designed a graphene-based optical refractive index sensor with high resolution of 1.7 × 10(-8) and sensitivity of 4.3 × 10(7) mV/RIU, as well as an extensive dynamic range. This highly sensitive graphene optical sensor enables label-free, live-cell, and highly accurate detection of a small quantity of cancer cells among normal cells at the single-cell level and the simultaneous detection and distinction of two cell lines without separation. It provides an accurate statistical distribution of normal and cancer cells with fewer cells. This facile and highly sensitive sensing refractive index may expand the practical applications of the biosensor.

A multishear microfluidic device for quantitative analysis of calcium dynamics in osteoblasts.

Microfluidics is a convenient platform to study the influences of fluid shear stress on calcium dynamics. Fluidic shear stress has been proven to affect bone cell functions and remodelling. We have developed a microfluidic system which can generate four shear flows in one device as a means to study cytosolic calcium concentration ([Ca(2+)](c)) dynamics of osteoblasts. Four shear forces were achieved by having four cell culture chambers with different widths while resistance correction channels compensated for the overall resistance to allow equal flow distribution towards the chambers. Computational simulation of the local shear stress distribution highlighted the preferred section in the cell chamber to measure the calcium dynamics. Osteoblasts showed an [Ca(2+)](c) increment proportional to the intensity of the shear stress from 0.03 to 0.30 Pa. A delay in response was observed with an activation threshold between 0.03 and 0.06 Pa. With computational modelling, our microfluidic device can offer controllable multishear stresses and perform quantitative comparisons of shear stress-induced intensity change of calcium in osteoblasts.

Optical trapping and manipulation of metallic micro/nanoparticles via photorefractive crystals

A simple method to trap and manipulate metallic micro/nano-particles on the surface of photorefractive crystals is proposed. After inducing inhomogeneous charge density and space-charge fields in photorefractive crystals by non-uniform illumination, both uncharged and charged metallic particles can be trapped on the illuminated surface due to dielectrophoretic force and electrophoretic force, respectively. A transition from dielectrophoresis to electrophoresis is observed when manipulating nano-silver particles with high surface space-charge field. Our results show that this method is simple and effective to form surface microstructures of metallic particles.

Elevation of extracellular Ca2+ induces store-operated calcium entry via calcium-sensing receptors: a pathway contributes to the proliferation of osteoblasts.

Aims The local concentration of extracellular Ca2+ ([Ca2+]o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca2+]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca2+]o to osteoblastic proliferation. Methods Cytosolic Ca2+ concentration ([Ca2+]c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail. Results Our data showed that elevating [Ca2+]o evoked a sustained increase of [Ca2+]c in a dose-dependent manner. This [Ca2+]c increase was blocked by TMB-8 (Ca2+ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca2+]o-induced [Ca2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists. Conclusions Elevating [Ca2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca2+ concentration.

Involvement of transient receptor potential melastatin-8 (TRPM8) in menthol-induced calcium entry, reactive oxygen species production and cell death in rheumatoid arthritis rat synovial fibroblasts.

Rheumatoid arthritis is most prominently characterized by synoviocyte hyperplasia which therefore serves as an important target for clinical therapy. In the present study, it was observed that menthol, the specific agonist of transient receptor potential melastatin subtype 8 (TRPM8), could induce sustained increases of cytosolic calcium concentration ([Ca(2+)]c) in synoviocytes isolated from collagen-induced arthritis rats in dose-dependent manner, which was evidently blocked by applying an extracellular Ca(2+)-free buffer. Menthol-induced [Ca(2+)]c increase was also significantly inhibited by potent TRPM8 antagonist capsazepine (CZP), indicating that this [Ca(2+)]c elevation was mostly attributed to TRPM8-mediated Ca(2+) entry. Besides, RT-PCR indeed demonstrated presence of TRPM8 in the synoviocytes. Meanwhile, it was found that menthol evoked production of intracellular reactive oxygen species, which could be abolished by Ca(2+) free solutions or CZP. Further experiments showed that menthol reduced the cell numbers and survival of synoviocytes. This reduction was associated with apoptosis as suggested by mitochondrial membrane depolarization, nuclear condensation and a caspase 3/7 apoptotic assay. Menthol-induced death and apoptosis of synoviocytes both were obviously inhibited by CZP, intracellular calcium chelator BAPTA-AM, and reactive oxygen species inhibitor diphenylene iodonium, respectively. Taken together, our data indicated that menthol resulted in synoviocyte death associated with apoptosis via calcium entry and reactive oxygen species production depending on TRPM8 activation.

Exogenous nitric oxide-induced release of calcium from intracellular IP3 receptor-sensitive stores via S-nitrosylation in respiratory burst-dependent neutrophils

PMA-induced respiratory burst neutrophils were exposed to exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) to study the effect of NO on calcium signaling. A sharp rise of cytosolic calcium concentration ([Ca(2+)](c)) was triggered by 1mM SNP with and without external calcium. We found that GF 109203X, a specific inhibitor of protein kinase C, DPI, a putative inhibitor of the respiratory burst-generating NADPH oxidase, and 2-DG, a non-metabolizable analog of glucose, completely inhibited the SNP-induced rise of [Ca(2+)](c) in PMA-activated respiratory burst neutrophils. Meanwhile, 2-APB and TMB-8, two potent IP(3) receptor inhibitors, prevented calcium increase respectively. Furthermore, N-ethylmaleimide (NEM), a specific cysteine alkylating agent, evidently abolished the [Ca(2+)](c) elevation. In contrast, the sGC inhibitor NS2028 had little effect on the rise of [Ca(2+)](c). Taken together, these results indicated that exogenous NO induced the release of calcium from intracellular IP(3) receptor-sensitive stores of neutrophils via S-nitrosylation in a respiratory burst-dependent manner.

Nitric oxide induces apoptosis associated with TRPV1 channel-mediated Ca2+ entry via S-nitrosylation in osteoblasts

The high-level production of nitric oxide (NO) induced by inflammatory cytokines has been shown to play a key role in the pathogenesis of inflammation-mediated osteoporosis. In the present work, we observed that 1mM of the NO donor sodium nitroprusside (SNP) induced an increase of the cytosolic calcium concentration ([Ca(2+)]c) in osteoblasts, which was completely abolished by applying an extracellular Ca(2+)-free buffer. Further experiments showed that the SNP-induced [Ca(2+)]c increase was specifically blocked by potent antagonists of the transient receptor potential vanilloid subtype 1 (TRPV1) channel: capsazepine, ruthenium red, and La(3+) in Ca(2+)-containing buffer. However, nifedipine, an L-type voltage sensitive Ca(2+)-channel blocker, failed to suppress the [Ca(2+)]c elevation caused by SNP. Additionally, 1mM SNP induced osteoblast apoptosis, which was largely inhibited by the blockers of TRPV1, capsazepine and ruthenium red. Interestingly, our data showed that the SNP-induced [Ca(2+)]c increase was significantly inhibited by N-ethylmaleimide, the blocker of S-nitrosylation modification, instead of inhibitors of the NO-cGMP-PKG pathway. Taken together, our data clearly demonstrated that the NO donor SNP resulted in apoptosis associated with TRPV1 channel-mediated Ca(2+) entry via S-nitrosylation in osteoblasts.

Ultraviolet irradiation induces autofluorescence enhancement via production of reactive oxygen species and photodecomposition in erythrocytes

Ultraviolet (UV) light has a significant influence on human health. In this study, human erythrocytes were exposed to UV light to investigate the effects of UV irradiation (UVI) on autofluorescence. Our results showed that high-dose continuous UVI enhanced erythrocyte autofluorescence, whereas low-dose pulsed UVI alone did not have this effect. Further, we found that H(2)O(2), one type of reactive oxygen species (ROS), accelerated autofluorescence enhancement under both continuous and pulsed UVI. In contrast, continuous and pulsed visible light did not result in erythrocyte autofluorescence enhancement in the presence or absence of H(2)O(2). Moreover, NAD(P)H had little effect on UVI-induced autofluorescence enhancement. From these studies, we conclude that UVI-induced erythrocyte autofluorescence enhancement via both UVI-dependent ROS production and photodecomposition. Finally, we present a theoretical study of this autofluorescence enhancement using a rate equation model. Notably, the results of this theoretical simulation agree well with the experimental data further supporting our conclusion that UVI plays two roles in the autofluorescence enhancement process.

Rhein antagonizes P2X7 receptor in rat peritoneal macrophages

P2X7 receptor plays important roles in inflammation and immunity, and thereby it serves as a potential therapeutic target for inflammatory diseases. Rhein, an anthraquinone derivative, exhibits significant anti-inflammatory and immunosuppressive activities in therapy. However, the underlying mechanisms are largely unclear. Here, we aimed to investigate the effects of rhein on P2X7 receptor-mediated responses in vitro. In HEK293 cells expressing rat P2X7 receptor, we first found that rhein concentration-dependently blocked ATP-induced cytosolic calcium concentration ([Ca2+]c) elevation and pore formation of the plasma membrane, two hallmarks of the P2X7 receptor activation. These two inhibitory effects of rhein were also observed in rat peritoneal macrophages. Furthermore, rhein counteracted macrophage phagocytosis attenuation and suppressed reactive oxygen species (ROS) production triggered by ATP/BzATP. Meanwhile, rhein reduced ATP/BzATP-induced IL-1β release in lipopolysaccharide-activated macrophages. Prolonged application of ATP caused macrophage apoptosis, while the presence of rhein suppressed this cell cytotoxicity. Such ATP/BzATP-induced cellular reactions were also inhibited by a well-known rat P2X7 receptor antagonist, brilliant blue G, in a similar way to rhein. Together, our results demonstrate that rhein inhibit ATP/BzATP-induced [Ca2+]c increase, pore formation, ROS production, phagocytosis attenuation, IL-1β release and cell apoptosis by antagonizing the P2X7 receptor in rat peritoneal macrophages.

Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.