Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

Background

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking.

Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

Highly sensitive detection of cytotoxicity using a modified HSP70B' promoter

We have previously found that the DNA fragment from nucleotides (nts) -287 to +110 in the HSP70B' gene is a functional promoter responding to Cadmium Chloride-induced cytotoxicity (Wada et al., Biotechnol Bioeng, 92, 410-415, 2005). In order to increase the cytotoxic response of this promoter, we first determined the location of the cytotoxic responding element (CRE) and then constructed tandem repeats of the CRE in front of the HSP70B' promoter. 5'- and 3'-deletion analysis revealed that the DNA fragment from nts -192 to -56 in the HSP70B' gene induces a significant response to cytotoxicity. When the AP-1 binding site in this region was mutated, the basal activity of HSP70B' gene promoter decreased but the cytotoxic response was unchanged. Thus, the CRE is located in nts -192 to -56 in the HSP70B' promoter, and the AP-1 binding site is not essential for the cytotoxic response. In addition, cells transfected with a luciferase construct carrying three tandem repeats of the CRE upstream of the HSP70B' promoter and containing AP-1 binding site mutation, showed a 2.28-fold higher response than that of no repeats. Moreover, the detection limit of Cadmium Chloride in the cells was 382 pmol/mL. Thus, highly sensitive sensor cells for Cadmium Chloride can be constructed using a HSP70B' promoter construct containing upstream tandem repeats of the CRE and mutation of the AP-1 binding site.     

L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells

Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G_q/11. In the present study we show that the Ca²⁺ current flowing through L-type voltage-gated Ca²⁺ channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca²⁺ channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Intraoperative detection of IDH-mutant glioma using fluorescence lifetime imaging

Identifying isocitrate dehydrogenase (IDH)-mutation and glioma subtype during surgery instead of days later can aid in modifying tumor resection strategies for better survival outcomes. We report intraoperative identification of IDH-mutant glioma (N = 12 patients) with a clinically compatible fluorescence lifetime imaging (FLIm) device (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm). The fluorescence-derived parameters were analyzed to study the optical contrast between IDH-mutant tumors and surrounding brain tissue. IDH-mutant oligodendrogliomas exhibited shorter lifetimes (3.3 ± 0.1 ns) than IDH-mutant astrocytomas (4.1 ± 0.1 ns). Both IDH-mutant glioma subtypes had shorter lifetimes than white matter (4.6 ± 0.4 ns) but had comparable lifetimes to cortex. Lifetimes also increased with malignancy grade within IDH-mutant oligodendrogliomas (grade 2: 2.96 ± 0.08 ns, grade 3: 3.4 ± 0.3 ns) but not within IDH-mutant astrocytomas. The current results support the feasibility of FLIm as a surgical adjuvant for identifying IDH-mutant glioma tissue.     

A reactive probe for Co2+ ion detection based on a catalytic decomposition process and its fluorescence imaging in living cells

A novel reactive fluorescent probe for cobalt ions was prepared based on integration of thiourea functional groups, coumarin, and naphthalimide fluorophores. There was no fluorescence observed for the probe itself, however, in the presence of cobalt ions, catalytic decomposition occurred for the probe and coumarin molecular fragments were produced that emitted blue fluorescence. This enabled the probe to be used as a ‘turn on’ reagent for detection of cobalt ions. Under physiological pH conditions and in appropriate solvent systems, an obvious fluorescence enhancement for cobalt ions was observed in selective experiments. Competition experiments indicated that cobalt ions could still induce fluorescence enhancement in the presence of other metal ions. Sensitivity experiments showed that the detection limit for cobalt ions was 6.0 nM. Dynamics research demonstrated that the catalytic process was a pseudo‐first‐order reaction and the reaction constant (k_obs) was calculated to be 1.49 × 10^?2 min^?1. In addition, the mechanism of catalytic decomposition could be demonstrated using electrospray ionization mass spectrometry and thin layer chromatography experiments. Cell fluorescence imaging experiments demonstrated that the probe could be used to detect cobalt ions in living HeLa cells.     

Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells

The Ca²⁺ and membrane binding protein annexin 2 can form a heterotetrameric complex with the S100A10 protein and this complex is thought to serve a bridging or scaffolding function in the membrane underlying cytoskeleton. To elucidate which of the subunits targets the complex to the subplasmalemmal region in live cells we employed YFP/CFP fusion proteins and live cell imaging in HepG2 cells. We show that monomeric annexin 2 is targeted to the plasma membrane whereas non-complexed S100A10 acquires a general cytosolic distribution. Co-expression of S100A10 together with annexin 2 and the resulting complex formation, however, lead to a recruitment of S100A10 to the plasma membrane thus identifying annexin 2 as the membrane targeting subunit.     

Highly sensitive fluorescence imaging of cancer with avidin-protease probe conjugate

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection.     

Real-time visualization of prion transport in single live cells using quantum dots

Prion diseases are fatal neurodegenerative disorders resulting from structural conversion of the cellular isoform of PrP^C to the infectious scrapie isoform PrP^Sc. It is believed that such structural alteration may occur within the internalization pathway. However, there is no direct evidence to support this hypothesis. Employing quantum dots (QDs) as a probe, we have recorded a real-time movie demonstrating the process of prion internalization in a living cell for the first time. The entire internalization process can be divided into four discrete but connected stages. In addition, using methyl-beta-cyclodextrin to disrupt cell membrane cholesterol, we show that lipid rafts play an important role in locating cellular PrP^C to the cell membrane and in initiating PrP^C endocytosis.     

Selective fluorescence sensing and quantification of uric acid by naphthyridine-based receptor in biological sample

Naphthyridine-based fluorescent probe H1 was synthesized and characterized for the quantification and selective detection of Uric Acid (UA) in live cell. In presence of UA, H1 forms the specific host-guest complex mainly through intermolecular hydrogen bonding and aromatic stacking which produces “turn-off” fluorescence. The probe and UA is found to be 1:1 stoichiometry on the basis of absorption and fluorescence titrations. The probe H1 has been shown to detect UA up to 0.6 µM at pH 7.4. DFT-TDDFT calculations were performed in order to demonstrate the sensing mechanism and the electronic properties of the receptor-donor complex. The selectivity was evaluated in Vero cells in the presence of UA with other purine derivatives, structurally similar to UA. It was found to exhibit no cytotoxicity effect in tested concentration of H1 and good membrane permeability for the detection of UA in living cell system. The unknown concentration of UA in serum and urine can be measured easily using the fluorescence property of probe H1.     

激光扫描共聚焦显微镜活细胞成像方法优化

激光扫描共聚焦显微镜可用于固定样品和活细胞样品的成像,近年来得到了广泛的应用。本文介绍了激光扫描共聚焦显微镜的基本原理及其在活细胞成像中的应用,并以FV10-ASW Viewer4.2软件为例,从扫描速度、分辨率、降噪、光电倍增调节、多参数协同优化、成像质量评估、图像后期处理等多个角度总结了激光扫描共聚焦活细胞成像系统的方法优化和推荐参数设置。本文的工作可以为活细胞实验提供一定参考。     

Highly effective colorimetric and visual detection of ATP by a DNAzyme-aptamer sensor

A new and simple method was developed to detect adenosine triphosphate (ATP) by using a DNAzyme aptamer sensor. The DNAzyme used was a single‐stranded DNA that could combine with hemin. The aptamer, a single, short nucleic acid sequence that can specifically bind with many targets, was an anti‐ATP aptamer. Two DNA sequences were designed: i) a functional chain (Chain A) consisting of two parts, i.e., the anti‐ATP aptamer (recognition part) and the DNAzyme (signal transduction part) and ii) a blocker chain (Chain B), which could partially hybridize with Chain A. The hybridized chains A and B were unfolded by the addition of ATP and hemin, and the blocker chain and the complex of the functional chain with ATP and hemin were in solution. The DNAzyme in the functional chain formed a G‐quadruplex with hemin and then catalyzed the oxidation by H₂O₂ of 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS²⁻) to the colored ABTS^.− radical. The color change caused by this reaction could be clearly observed by naked eye, and the absorbance was recorded at 414 nm. The detection limit was 1×10⁻⁶ M .     

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.     

Improved detection of apoptotic cells using a modified in situ TUNEL technique.

We are in the process of assessing the response of cancer tissues to chemotherapy, evaluating, among other points, the proportion of cancer cells undergoing apoptosis. However, the apoptotic index obtained with the original TUNEL technique was lower than that obtained by evaluation of apoptosis on H&E-stained sections. Here we describe a small modification of the TUNEL technique that significantly increases the sensitivity of the assay. In the nonmodified TUNEL technique, a digoxigenin-labeled probe is detected using a direct peroxidase-conjugated system, whereas here we report the advantage of using a streptavidin-biotin-immunoperoxidase system. This, in conjunction with pretreatment of tissue sections with proteinase K and microwave irradiation, improved the detection of apoptotic cells.     

In vivo MR imaging of magnetically labeled human embryonic stem cells

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.     

The PDZ-LIM protein RIL modulates actin stress fiber turnover and enhances the association of alpha-actinin with F-actin

ALP, CLP-36 and RIL form the ALP subfamily of PDZ-LIM proteins. ALP has been implicated in sarcomere function in muscle cells in association with alpha-actinin. The closely related CLP-36 is predominantly expressed in nonmuscle cells, where it localizes to actin stress fibers also in association with alpha-actinin. Here we have studied the expression and functions of RIL originally identified as a gene downregulated in H-ras-transformed cells. RIL was mostly expressed in nonmuscle epithelial cells with a pattern distinct from that of CLP-36. RIL protein was found to localize to actin stress fibers in nonmuscle cells similarly to CLP-36. However, RIL expression led to partially abnormal actin filaments showing thick irregular stress fibers not seen with CLP-36. Furthermore, live cell imaging demonstrated altered stress fiber dynamics with rapid formation of new fibers and frequent collapse of thick irregular fibers in EGFP-RIL-expressing cells. These effects may be mediated through the association of RIL with alpha-actinin, as RIL was found to associate with alpha-actinin via its PDZ domain, and RIL enhanced the ability of alpha-actinin to cosediment with actin filaments. These results implicate the RIL PDZ-LIM protein as a regulator of actin stress fiber turnover.     

Highly sensitive detection of cocaine using a piezoelectric immunosensor

This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.