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Newly emerging super-resolution imaging techniques provide opportunities for precise observations on cellular microstructures. However, they also impose severe demands on fluorophores. Here, we develop a new series of NIR xanthene dyes, named as KRhs, by replacing the 10-position O of rhodamines with a cyclo-ketal. KRhs display an intense NIR emission peak at 700 nm with fluorescence quantum yields up to 0.64. More importantly, they, without the aid of enhancing buffer, exhibit stochastic fluorescence off-on switches to support time-resolved localization of single fluorophore. KRhs are functionalized into KRh-MitoFix, KRh-Mem and KRh-Halo that demonstrate mitochondria, plasma membrane and fusion protein targeting ability, respectively. Consequently, these KRh probes demonstrate straightforward usage for super-resolution imaging of these targets in live cells. Therefore, KRhs merit future development for fluorescence labeling and super-resolution imaging in the NIR region., (© 2020 Wiley-VCH GmbH.)

Actinobacteria are one of the most promising producers of medically and industrially relevant secondary metabolites. However, screening of such compounds in actinobacteria growth demands simple, fast, and efficient extraction procedures that enable detection and precise quantification of biologically active compounds. In this regard, solid phase microextraction (SPME) emerges as an ideal extraction technique for screening of secondary metabolites in bacteria culture due to its non-exhaustive, minimally invasive, and non-destructive nature: its integrated sample preparation workflow; balanced coverage feature; metabolism quenching capabilities; and superior cleanup, as well as its versatility in configuration, which enables automation and high throughput applications. The current work provides a comparison of micro-scale and direct immersion SPME (DI-SPME) for screening of secondary metabolites, describes the optimization of the developed DI-SPME method, and introduces the developed technique for mapping of target secondary metabolites as well as its direct coupling to mass spectrometry for such applications. The optimized DI-SPME method provided higher amounts of extracted ions and intensity signals, yielding superior extraction and desorption efficiency as compared with micro-scale extraction. Studied compounds presented stability on the coating for 24 h at room temperature. The DI-SPME mapping approach revealed that lysolipin I and the lienomycin analog are distributed along the center and edges of the colony, respectively. Direct coupling of SPME to MS provided a similar ions profile as SPME-LC-MS while enabling a significant decrease in analysis time, demonstrating its suitability for such applications. DI-SPME is herein presented as an alternative to micro-scale extraction for screening of secondary metabolites in actinobacteria solid medium, as well as a feasible alternative to DESI-IMS for mapping of biologic radial distribution of secondary metabolites and cell life cycle studies. Lastly, the direct coupling of DI-SPME to MS is presented as a fast, powerful technique for high throughput analysis of secondary metabolites in this medium., (© 2019 John Wiley & Sons, Ltd.)

Ergot, fungal genus Claviceps , are worldwide distributed grass pathogens known for their production of toxic ergot alkaloids (EAs) and the great agricultural impact they have on both cereal crop and farm animal production. EAs are traditionally considered as the only factor responsible for ergot toxicity. Using broad sampling covering 13 ergot species infecting wild or agricultural grasses (including cereals) across Europe, USA, New Zealand, and South Africa we showed that the content of ergochrome pigments were comparable to the content of EAs in sclerotia. While secalonic acids A-C (SAs), the main ergot ergochromes (ECs), are well known toxins, our study is the first to address the question about their contribution to overall ergot toxicity. Based on our and published data, the importance of SAs in acute intoxication seems to be negligible, but the effect of chronic exposure needs to be evaluated. Nevertheless, they have biological activities at doses corresponding to quantities found in natural conditions. Our study highlights the need for a re-evaluation of ergot toxicity mechanisms and further studies of SAs' impact on livestock production and food safety.

In vitro assays (such as resazurin and MTT) provide an opportunity to determine the cytotoxicity of novel therapeutics before moving forward with expensive and resource-intensive in vivo studies. A concern with using these assays, however, is the production of false responses in the presence of particular chemical functionalities. To better understand this phenomenon, 19 small molecules at 6 concentrations (1 μM-100 mM) were tested in the presence of resazurin and MTT reagents to highlight potential interfering species. Through the use of absorbance measurements (using well-plate assays and UV-vis spectroscopy) with parallel MS analysis, we have shown that significant conversion of the assay reagents readily occurs in the presence of many tested interfering species without the need for any cellular activity. The most attributable sources of interference seem to arise from the presence of thiol and carboxylic acid moieties. Interestingly, the detectable interferences were more prevalent and larger in the presence of MTT (19 species with some deviations >3000%) compared to resazurin (16 species with largest deviation of ∼150%). Additionally, those deviations in the presence of resazurin were only substantial at high concentrations, while MTT showed deviations across the tested concentrations. This comprehensive study gives insight into chemical functional groups (thiols, amines, amides, carboxylic acids) that may interfere with resazurin and MTT assays in the absence of metabolic activity and indicates that proper control studies must be performed to obtain accurate data from these in vitro assays.

Saposhnikoviae Radix ("Boufu") is an important crude drug used in Kampo formulation. It is extracted from wild-type plants. However, recently, extraction has become difficult because of a decrease in wild-type plants. Therefore, cultivated plants account for the majority of the market, from which the crude drug is extracted. However, the cultivation techniques used are not sufficient to obtain the desirable extracts. In this study, we compared the contents of the extract and the quantitative values of characteristic constituents obtained from wild-type and cultivated plants, and found a remarkable difference. Therefore, it is considered that these indicators play an important role in the establishment of better cultivation technology.

Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non-fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram-positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration, and pre-exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram-positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity., (© 2017 Wiley Periodicals, Inc.)

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Streptococcus pneumoniae is a major human pathogen, causing around 1.6 million deaths worldwide each year. By optimizing a resazurin-based assay to detect S. pneumoniae growth in 384-well microplates, we developed a new high-throughput screening (HTS) system for the discovery of antipneumococcal molecules, which was unsuccessful using conventional absorbance measurements. Before applying our protocol to a large-scale screen, we validated the system through a pilot screen targeting about 7,800 bioactive molecules using three different S. pneumoniae serotypes. Primary screenings of a further 27,000 synthetic small molecules facilitated the identification of 3-acyl-2-phenylamino-1,4-dihydropquinolin-4-one (APDQ) derivatives that inhibited growth of S. pneumoniae with MIC 90 values <1 μM (0.03-0.81 μM). Five selected APDQ derivatives were also active against Staphylococcus aureus but neither Klebsiella pneumoniae nor Pseudomonas aeruginosa, suggesting that APDQ may act specifically against Gram-positive bacteria. Our results both validated and demonstrated the utility of the resazurin-based HTS system for the identification of new antipneumococcal molecules. Moreover, the identified new antipneumococcal molecules in this study may have potential to be further developed as new antibiotics.

Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC 90 values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals., (Copyright © 2017 American Society for Microbiology.)

The budding yeast S. cerevisiae is widely used as a eukaryotic model organism to elucidate the mechanism of action of low molecular weight compounds. This report describes the development of two high throughput screening methods based on cell viability either by monitoring the reduction of alamarBlue ® (resazurin) or by direct optical measurement of cell growth. Both methods can be miniaturized to allow screening of large numbers of samples, and can be performed using S. cerevisiae in 384 and 1536-well format. The alamarBlue ® approach achieves Z' values of >0.7 with signal to basal ratios of >6.5, and around 1.1 million low molecular weight compounds were screened, identifying approximately 25,000 primary hits. Dose response curves generated for a subset (1930) using both alamarBlue ® and optical density methods showed significant overlap. In genome-wide haploinsufficiency profiling (HIP), 572 of these hits demonstrated a diverse mechanism of action, affecting >25% of all yeast strains., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Resazurin microtitre assay (RMA) has been successfully used to detect minimal inhibitory concentrations (MICs) of both first-line and several second-line drugs in drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). In this study, we firstly compared prothionamide (PTH) susceptibility testing of Mycobacterium tuberculosis (MTB) using resazurin microtitre assay (RMA) and MGIT. Overall, the sensitivity and specificity of RMA for detecting PTH susceptibility was 96.5% [95% confidence interval (CI): 91.7-100.0] and 93.2% (95% CI: 89.6-96.8) respectively. In addition, the median time to positivity was significantly shorter for RMA than for the automated MGIT 960 (RMA, 8 days [range: 8-8 days] vs MGIT, 10.1 days, [range: 5.0-13.0]; P < 0.01). Concordance rate for MICs between RMA and MGIT for PTH-resistant group was 64.3% (95% CI: 46.5-82.0), which was significantly lower than that of PTH-susceptible group (85.9%, 95% CI: 78.8-93.0; P= 0.01). In conclusion, our data demonstrated that RMA can be used as an acceptable alternative for determination of PTH susceptibility with shorter turn-around time. When compared with MGIT 960, RMA method was prone to produce higher MICs for PTH-resistant MTB strains.

Primary cultured cardiomyocytes show spontaneous Ca 2+ oscillations (SCOs) which not only govern contractile events, but undergo derangements that promote arrhythmogenesis through Ca 2+ -dependent mechanism. We systematically examined influence on SCOs of an array of ion channel modifiers by recording intracellular Ca 2+ dynamics in rat ventricular cardiomyocytes using Ca 2+ specific fluorescence dye, Fluo-8/AM. Voltage-gated sodium channels (VGSCs) activation elongates SCO duration and reduces SCO frequency while inhibition of VGSCs decreases SCO frequency without affecting amplitude and duration. Inhibition of voltage-gated potassium channel increases SCO duration. Direct activation of L-type Ca 2+ channels (LTCCs) induces SCO bursts while suppressing LTCCs decreases SCO amplitude and slightly increases SCO frequency. Activation of ryanodine receptors (RyRs) increases SCO duration and decreases both SCO amplitude and frequency while inhibiting RyRs decreases SCO frequency without affecting amplitude and duration. The potencies of these ion channel modifiers on SCO responses are generally consistent with their affinities in respective targets demonstrating that modification of distinct targets produces different SCO profiles. We further demonstrate that clinically-used drugs that produce Long-QT syndrome including cisapride, dofetilide, sotalol, and quinidine all induce SCO bursts while verapamil has no effect. Therefore, occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing Long-QT syndrome.

Confocal microscopy, coupled to high-performance hardware and software systems, has provided scientists with the capability of overcoming some of the limitations of standard microscopic imaging measurements of intracellular ions. The technique for loading of ion fluorescent probes is easily achieved; however, the quality of calcium measurements depends on the way of using the confocal system. In order to optimize this technique, scientists need to be familiar with the basic approaches and limitations of confocal microscopy. In this chapter, we will describe sample preparation, fluorescent probe loading, labeling of intracellular compartments, and the setting of parameters as well as protocols for measurements and limitations of the technique.

Extraction and clean-up methods were examined for the analysis of acidic tar dyes in various high-protein foods. 1% Aqueous ammonia followed by ethanol, 1% aqueous ammonia-ethanol (1 : 1) mixture, and 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture were used in sequence for boiled fish paste (kamaboko), pounded fish cake (hanpen), and sausage. The sausage extract was centrifuged at low temperature to solidify and remove the contained fat. Salted cod roe with red pepper was extracted twice with 1% aqueous ammonia-ethanol (1 : 1) mixture, followed by extraction with 1% aqueous ammonia-tetrahydrofuran (1 : 1) mixture. A divinylbenzene-N-vinylpyrrolidone copolymer column was used for the clean-up of xanthen dyes. In the case of clogging-prone samples, the same type of large-particle-size column was used. A polyamide column was used for clean-up of the other dyes. When each dye was added at 5 μg/g in the foods, recoveries from kamaboko, hanpen, and sausage ranged from 76 to 102%, and the average recovery from the two types of salted cold roe with red pepper ranged from 45 to 98%.

We report the in vitro drugs interaction by the resazurin drugs combination microtiter assay (REDCA) of amoxicillin (AMO)/clavulanate (CLAV) with isoniazid (INH), ethambutol (EMB), and rifampicin (RIF) against susceptible and resistant Mycobacterium tuberculosis isolates. The addition of AMO/CLAV to classical antituberculosis drugs should be explored as a promising alternative for the treatment of resistant tuberculosis (TB)., (Copyright © 2015. Published by Elsevier B.V.)

Herein, we report a novel lysosome targetable luminescent bioprobe derived from a europium coordination compound, namely Eu(pfphOCH 3 IN) 3 (DDXPO) 4 [where HpfphOCH 3 IN = 4,4,5,5,5-pentafluoro-3-hydroxy-1-(1-(4-methoxyphenyl)-1H-indol-3-yl)pent-2-en-1-one and DDXPO = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene oxide]. Notably, the newly designed europium complex exhibits significant quantum yield (Φ overall = 25 ± 3%) and 5 D 0 excited state lifetime (τ = 398 ± 3 μs) values under physiological pH (7.2) conditions when excited at 405 nm. Hence the developed europium complex has been evaluated for live cell imaging applications using mouse pre-adipocyte cell lines (3T3L1). Colocalization studies of the designed bio-probe with commercial Lysosome-GFP in 3T3L1 cells demonstrated the specific localization of the probe in the lysosome with a high colocalization coefficient (A = 0.83). Most importantly, the developed bioprobe exhibits good cell permeability, photostability and non-cytotoxicity.

In this work, two xanthene dyes (H-hNR and TF-hNR) have been synthesized by a convenient and efficient method. These two dyes exhibited deep-red and near-infrared emissions, high fluorescence quantum yields, and good photostability. Their structure-optical properties were investigated by X-ray crystal structure analysis and density functional theory calculations. Live cell imaging data revealed that H-hNR and TF-hNR could rapidly stain both A549 and HeLa cells with low concentrations. The excellent photophysical and imaging properties render them as promising candidates for use in live cell imaging.

Objectives: The objectives of this study were to: (i) determine the in vitro activities of a series of di-, tri- and tetra-nuclear ruthenium complexes (Rubbn, Rubbn-tri and Rubbn-tetra) against a range of Gram-positive and -negative bacteria and compare the antimicrobial activities with the corresponding toxicities against eukaryotic cells; and (ii) compare MIC values with achievable in vivo serum concentrations for the least toxic ruthenium complex., Methods: The in vitro activities were determined by MIC assays and time-kill curve experiments, while the toxicities of the ruthenium complexes were determined using the Alamar blue cytotoxicity assay. A preliminary pharmacokinetic study was undertaken to determine the Rubb12 serum concentration in mice as a function of time after administration., Results: Rubb12, Rubb12-tri and Rubb12-tetra are highly active, with MIC values of 1-2 mg/L (0.5-1.5 μM) for a range of Gram-positive strains, but showed variable activities against a panel of Gram-negative bacteria. Time-kill experiments indicated that Rubb12, Rubb12-tri and Rubb12-tetra are bactericidal and kill bacteria within 3-8 h. The di-, tri- and tetra-nuclear complexes were ∼50 times more toxic to Gram-positive bacteria and 25 times more toxic to Gram-negative strains, classified as susceptible, than to liver and kidney cells. Preliminary pharmacokinetic experiments established that serum concentrations higher than MIC values can be obtained for Rubb12 with an administered dose of 32 mg/kg., Conclusions: The ruthenium complexes, particularly Rubb12, have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complex can be readily further modified in order to increase the selectivity for bacteria over eukaryotic cells., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid., Methods: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation., Results: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid., Conclusions: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Due to the advancements in modern medicine that have resulted in an increased number of immunocompromised individuals, the incidences and the associated mortality of invasive aspergillosis have continued to rise over the past three decades despite appropriate treatment. As a result, invasive aspergillosis has emerged as a leading cause of infection-related mortality in immunocompromised individuals. Utilizing the resazurin to resorufin conversion fluorescence readout to monitor cell viability, herein, we outline a high-throughput screening method amenable to profiling a large pharmaceutical library against the clinically relevant but less frequently screened fungal pathogen Aspergillus fumigatus. This enables the user to conduct high-throughput screening using a disease-relevant fungal growth assay and identify novel antifungal chemotypes as drug leads.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages., (© 2015 International Society for Advancement of Cytometry.)

Background: The resazurin microtiter assay (classic REMA), a colorimetric liquid culture-based drug susceptibility assay for Mycobacterium tuberculosis (MTB), has been endorsed by the World Health Organization. The assay requires 8-16 days to obtain results, delaying management of drug resistant tuberculosis patients. A modified REMA which allows results in as little as 24 hours for bacterial strains, has been developed and validated using Staphylococcus aureus, but has not yet been evaluated for MTB. Therefore we assessed the performance of the modified REMA for rifampicin (RIF) and isoniazid (INH) susceptibility, using the classic REMA as the reference standard. We also compared simplicity (from the technicians' point of view), time taken to obtain results (rank-sum testing), specificity and Kappa statistics of the two methods., Results: The modified REMA, which is a one-step procedure, was found to be simpler to perform and results were obtained in a significantly shorter time (5 versus 9 days, p < 0.0001) compared to the classic REMA due to addition of indicator and strain at the same time. The specificity of the modified REMA was low {46.8% (35.5% - 58.4%) for RIF and 13.9% (7.2% - 23.5%) for INH}. Kappa statistics were 16.0% for RIF and 2.0% for INH. Low specificity and kappa statistics are due to indicator reduction by the strains before complete drug activity., Conclusion: Although modified REMA is faster and simpler compared to classic REMA, it is not reliable for MTB drug susceptibility testing.

Aims: Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca., Methods and Results: Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s(-1). Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased., Conclusions: This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.)

We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: To facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes., Methods: Based on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum., Results: Both uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++., Conclusions: Nondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

Resazurin (RNO) reduction by hydrated electrons produces a fluorescent molecule: resorufin (RN). To take advantage of RN fluorescence, a novel setup is designed by implementing fluorescence detection induced by laser in a pulse radiolysis experiment. Time resolved fluorescence spectra were recorded with a fast gated intensified CCD camera during the reduction of RNO from μs to ms. Two 532 nm laser types have been used to describe the short μs range by a 5 ns Q-switch laser and the μs-ms range by a CW DPSS laser. By fitting the simulated model to the experimental data a second order rate constant of 10(9) M(-1) s(-1) was re-evaluated. This method should be considered in the near future in many in situ and real time measurements for evaluating radical production.

Introduction: Although adenosine triphosphate (ATP) has often been reported to relax the corpus cavernosum, this may be mediated by indirect effects, such as release of nitric oxide from the endothelium. Recent data suggest that P2X(1) receptors may be up-regulated in diabetes, and these exert an anti-erectile effect by causing the corpus cavernosum smooth muscle cells (CCSMCs) to contract. However, to date, there is no functional evidence that ATP can directly stimulate CCSMC., Aims: This study aims to (i) to directly examine the effect of ATP on membrane currents in freshly isolated CCSMC, where influences of endothelium and other cells are absent; and (ii) to determine the receptor subtypes, ionic currents, and Ca(2+) signals stimulated by ATP., Methods: CCSMCs were enzymatically dispersed from male New Zealand White rabbits for patch clamp recording and measurement of intracellular Ca(2+) in fluo-4-loaded cells using spinning disk confocal microscopy., Main Outcome Measures: Patch clamp recordings were made of ATP-evoked membrane currents and spontaneous membrane currents. Spinning disk confocal imaging of intracellular Ca(2+) was performed, and the response to ATP was recorded., Results: ATP evoked repeatable inward currents in CCSMC (1st application: -675 ± 101 pA; 2nd application: -694 ± 120 pA, N = 9, P = 0.77). ATP-induced currents were reduced by suramin from -380 ± 121 to -124 ± 37 pA (N = 8, P < 0.05), by α,β-methylene ATP from -755 ± 235 to 139 ± 49 pA (N = 5, P < 0.05), and by NF449 from -419 ± to -51 ± 13 pA (N = 6, P < 0.05). In contrast, MRS2500, a P2Y1(1,12,13) antagonist, had no effect on ATP responses (control: -838 ± 139 pA; in MRS2500: -822 ± 184 pA, N = 13, P = 0.84) but blocked inward currents evoked by 2-MeSATP, a P2Y1,12,13 agonist (control: -623 ± 166 pA; in MRS2500: -56 ± 25 pA, N = 6, P < 0.05). The ATP-evoked inward current was unaffected by changing the transmembrane Cl(-) gradient but reversed in direction when extracellular Na(+) was reduced, indicating that it was a cation current., Conclusions: ATP directly stimulates CCSMC by evoking a P2X-mediated cation current., (© 2013 International Society for Sexual Medicine.)

To simultaneously determine paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills. In the test, Insertil ODS-C18 column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the column temperature was 30 degrees C and the detection wavelength was 230 nm. According to the results of the test, paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol showed good linear relations between peak areas and sample sizes in 11.37-170.5, 2.188-32.82, 2.896-43.44, and 3.000-45.00 mg x L(-1) (r = 0.999 9, 0.999 9, 1.000 0, 1.000 0), respectively. The average recoveries (n = 6) were 102.3% (RSD 1.2%), 99.71% (RSD 1.9%), 101.2% (RSD 1.2%), and 99.40% (RSD 2.4%), respectively. The above four components were determined in five batches of samples by using the established method, with satisfactory results. The method was so simple, accurate and highly reproducible that it could be used for quality control of the four components in Zhengtian pills.

Pyrazinamide (PZA) is a first-line antituberculosis (anti-TB) drug capable of killing nonreplicating, persistent Mycobacterium tuberculosis. However, reliable testing of the susceptibility of M. tuberculosis to PZA is challenging. Using 432 clinical M. tuberculosis isolates, we compared the performances of five methods for the determination of M. tuberculosis susceptibility to PZA: the MGIT 960 system, the molecular drug susceptibility test (mDST), the pyrazinamidase (PZase) activity assay, the resazurin microtiter assay (REMA), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction test. The sensitivities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA, and the MTT assay were 98.8%, 88.8%, 90.5%, 98.8%, and 98.2%, respectively. The sensitivities of the PZase activity assay and the mDST were lower than those of the other three methods (P < 0.05). The specificities of the MGIT 960 system, the PZase activity assay, the mDST, the REMA and the MTT assays were 99.2%, 98.9%, 90.9%, 98.5%, and 100%, respectively. The specificity of the mDST was lower than those of the other four methods (P < 0.05). In conclusion, the MGIT 960 system, the MTT assay, and the REMA are superior to the PZase activity assay and the mDST in determining the susceptibility of M. tuberculosis to PZA. The MTT assay and the REMA might serve as alternative methods for clinical laboratories without access to the MGIT 960 system. For rapid testing in well-equipped laboratories, the mDST might be the best choice, particularly for small quantities of M. tuberculosis. The PZase activity assay has no obvious advantage in the assessment of M. tuberculosis susceptibility to PZA, as it is less accurate and requires larger quantities of bacteria.

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)(1,2,3). Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis(4). These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability(5). Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis(6). The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported(2). These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

The circling (cir/cir) mouse is a spontaneous model of deafness due to deletion of a 40-kb genomic region that includes the transmembrane inner ear (tmie) gene. In addition to being deaf, cir/cir mice exhibit abnormal behaviors including circling and hyperactivity. Here we investigated differences between 3-d-old (that is, before hair-cell degeneration) cir/cir and phenotypically normal (+/cir) mice and the reason underlying the degeneration of the inner ear structure of cir/cir mice. To this end, we used gentamicin, gentamicin-Texas red conjugate, and FM1-43 to investigate mechanotransducer channel activity in the hair cells of cir/cir mice; these compounds are presumed to enter hair cells through the mechanotransducer channel. Although the structure of the inner ear of +/cir mice was equivalent to that of cir/cir mice, the hair cells of cir/cir mice (unlike +/cir) did not take up gentamicin, gentamicin-Texas red conjugate, or FM1-43. These findings suggest that hair cells in cir/cir mice demonstrate abnormal maturation and mechanotransduction. In addition, our current results indicate that tmie is required for maturation and maintenance of hair cells.

In this study, metabolite profiling of Radix Saposhnikoviae from different geographical locations was performed using high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOFMS) and multivariate statistical analysis technique. Principle component analysis (PCA) of the data shows that these samples could be roughly separated into three groups: Guan Fangfeng, Kou Fangfeng and Chuan Fangfeng. The potential chemical markers were discovered through the loading plot of PCA. Based on accurate mass measurements and subsequent fragment ions of TOFMS after in-source collision induced dissociation, as well as matching of empirical molecular formulae with those of published components in the in-house chemical library, 10 potential markers, such as 4'-O-glucosyl-5-O-methylvisamminol, cimifugin, prim-O-glucosylcimifugin and 3'-O-angeloylhammaudol, were tentatively identified and partially verified by the available reference standards. The results of this study indicate that it is an effective and novel approach to identify traditional Chinese medicine (TCM) from different sources, and that performing quantity determination of corresponding marker compounds could optimize the quality control of TCM.

The antibiotic rifampicin is widely used in the treatment of tuberculosis. Side effects of rifampicin include hemolytic anemia. Loss of circulating erythrocytes resembling hemolytic anemia could result from stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)) and formation of ceramide. The present study explored, whether and, if so, how rifampicin triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, cell volume from forward scatter in flow cytometry, PS exposure from annexin binding, ceramide formation from binding of fluorescent antibodies and hemolysis from hemoglobin release. As a result, a 48 h exposure to rifampicin (≥ 24 μg/ml) significantly increased Fluo3 fluorescence, ceramide abundance and annexin binding, and significantly decreased forward scatter. Rifampicin triggered slight, but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not fully abolish rifampicin induced annexin binding. In conclusion, exposure of human erythrocytes to rifampicin is followed by suicidal erythrocyte death or eryptosis, an effect at least partially due to increase of cytosolic Ca(2+) concentration and stimulation of ceramide formation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Side effects of gossypol, a polyphenolic component of Gossypium, with male contraceptive, anticancer, antimicrobial and antiviral activities include anemia due to accelerated demise of erythrocytes. Erythrocytes may be cleared from circulating blood following apoptosis-like suicidal death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether gossypol stimulates eryptosis of human erythrocytes. Utilizing flow cytometry, [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. A 48 h exposure to gossypol (0.75 μM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Gossypol exposure was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) significantly blunted the effect of gossypol (1 μM) on annexin-V-binding. The present observations reveal a novel effect of gossypol on human erythrocytes, which contributes to or even accounts for the triggering of anemia by this substance., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Aims: The expression of cell adhesion molecules on vascular smooth muscle cells is central to leukocyte recruitment and progression of atherosclerotic disease. Ohioensin F, a chemical compound of the Antarctic moss Polyerichastrum alpinum, exhibited inhibitory activity against protein tyrosine phosphatase 1B and antioxidant activity. However, published scientific information regarding other biological activities and pharmacological function of ohioensin F is scarce. In the present study, we aimed to examine the in vitro effects of ohioensin F on the ability to suppress TNF-α-induced adhesion molecule expression in vascular smooth muscle cells (VSMCs)., Main Methods: The inhibitory effect of ohioensin F on TNF-α-induced upregulation in expression of adhesion molecules was investigated by enzyme-linked immunosorbent assay, cell adhesion assay, RT-PCR, western blot analysis, immunofluorescence, and transfection and reporter assay, respectively., Key Findings: Pretreatment of VSMCs with ohioensin F at nontoxic concentrations of 0.1-10 μg/ml dose-dependently inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). In addition, ohioensin F suppressed adhesion of THP-1 monocytes to TNF-α-stimulated VSMCs. Ohioensin F reduced TNF-α-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Finally, ohioensin F inhibited TNF-α-induced CAM mRNA expression and NK-κB translocation., Significance: These results suggest a new mechanism of ohioensin F's anti-inflammatory action, owing to the negative regulation of TNF-α-induced adhesion molecule expression, monocyte adhesion and ROS production in vascular smooth muscle cells. Our finding also supports ohioensin F as a potential pharmacological, anti-inflammatory molecule that has a protective effect on the atherosclerotic lesion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Preliminary data suggest different intracellular calcium handling of Th1 and Th2 lymphocytes that may contribute to distinct cytokine production patterns. In this study we explored the contribution of the main mechanisms in charge of the elevation and decrease of cytoplasmic free calcium levels, i.e., the endoplasmic calcium release, the calcium release activated calcium (CRAC) channel, the mitochondrial calcium uniporter (MCU), the sarco/endoplasmic reticulum calcium ATPase (SERCA), and the plasma membrane calcium ATPase (PMCA) during the first 10 min of activation in human Th1 and Th2 lymphocytes applying a kinetic flow cytometry approach. We isolated peripheral blood mononuclear cells from 10 healthy individuals. Cells were stained with CD4, CXCR3 and CCR4 cell surface markers to identify Th1 and Th2 cells, respectively and loaded with Fluo-3/AM calcium sensitive dye. Cells were activated with phytohemagglutinine and alterations of cytoplasmic free calcium levels were monitored for 10 min after specific inhibition of the above mechanisms. Our results revealed delicate differences in calcium flux kinetics of Th1 and Th2 lymphocytes. The lower activity of MCU, and therefore of CRAC channels, along with the higher activity of the SERCA pump account for the notion that Th2 cells go through a lower level of lymphocyte activation compared with Th1 cells upon identical activating stimuli. The observed differences in calcium flux of Th1 and Th2 cells may contribute to different calcium handling kinetics and, hence, to distinct cytokine production patterns by these subsets., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis., Methods: [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies., Results: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca(2+)](i), enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding., Conclusion: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure., (Copyright © 2012 S. Karger AG, Basel.)

Background: Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available., Aim: This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates., Methods: A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test., Results: Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days., Conclusion: Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background/aims: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown., Methods: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin., Results: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways., Conclusions: We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process., (Copyright © 2012 S. Karger AG, Basel.)

Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays; however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (~10 μm). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion., (© 2011 American Chemical Society)

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca2+ entry and neuronal Ca2+ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K+-evoked [Ca2+]c transients with an IC50 of 5.31 μM. At 10 μM, the compound decreased the amplitude and area of the Ca2+ transient elicited by challenging single fura-2-loaded cells with high K+, by 40% and 80%, respectively. This concentration also caused a blockade of K+-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca2+ currents (IC50=6.52 μM). At 10 μM, ITH/IQM also inhibited the Ca2+-dependent outward K+ current, leaving untouched the voltage-dependent component of IK. The inward Na+ current was unaffected. Inhibition of depolarisation-elicited Ca2+ entry, [Ca2+]c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine (RGDS)-coupled calcium alginates (1.2 percent, w/v), allowed to attach to the substratum and then cryopreserved in 10 percent (v/v) dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1 C per min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR) and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93 ± 2 percent, mean and standard error) than those in suspension (52 ± 2 percent) or in unmodified alginates (62 ± 3 percent). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide per g alginate, 30 minutes loading time and 1 C per min cooling rate.

An efficient solid phase strategy for the versatile functionalisation of xanthene dyes for conjugation and labelling of biomolecules is presented. The low cost, high purity and excellent spectral properties of the obtained materials provide an attractive alternative for the labelling of a wide range of molecules.

One of the problems in imaging from brain tissues is light-scattering. Thus, multiphoton laser scanning microscopy is widely used to optically access fluorescent signals located deeply in tissues. Here we report that Nipkow-type spinning-disk one-photon confocal microscopy, which embodies high temporal resolution and slow photobleaching, is also capable of imaging tissues to a depth of up to 150 μm. Using a Nipkow-disk microscope, we conducted functional multi-cell calcium imaging of CA3 neurons from in toto intact hippocampal preparations and astrocytes from in vivo neocortical layer 1. This novel application of Nipkow-disk microscopy expands the potential usefulness of this type of microscopy and will contribute to our understanding of natural neuronal microcircuitry., (© Imperial College Press)

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\tandem mass spectrometry (HPLC-MS(n)) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC-ESI-MS(n). Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin (2) and 4'-O-β-D-glucosyl-5-O-methylvisamminol (4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin (1), 5-O-methylvisamminol (3) and sec-O-glucosylhamaudol (5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC-ESI-MS(n) for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aims: N-n-Butyl haloperidol iodide (F(2)) is a novel compound derived from haloperidol. In our previous work, F(2) was found to be an L-type calcium channel blocker which played a protective role in rat heart ischemic-reperfusion injury in a dose-dependent manner. In the current study, we aimed to investigate the effects and some possible mechanisms of F(2) on calcium transients in hypoxic/ischemic rat cardiac myocytes., Methods and Results: Calcium transients' images of rat cardiac myocytes were recorded during simulated hypoxia, using a confocal calcium imaging system. The amplitude, rising time from 25% to 75% (RT25-75), decay time from 75% to 25% (DT75-25) of calcium transients, and resting [Ca(2+)](i) were extracted from the images by self-coding programs. In this study, hypoxia produced a substantial increase in diastolic [Ca(2+)](i) and reduced the amplitude of calcium transients. Both RT25-75 and DT75-25 of Ca(2+) transients were significantly prolonged. And F(2) could reduce the increase in resting [Ca(2+)](i)and the prolongation of RT25-75 and DT75-25 of Ca(2+) transients during hypoxia. F(2) also inhibited the reduction in amplitude of calcium transients which was caused by 30-min hypoxia. The activity of SERCA2a (sarcoplasmic reticulum Ca(2+)-ATPase, determined by test kits) decreased after 30-min ischemia, and intravenous F(2) in rats could ameliorate the decreased activity of SERCA2a. The inward and outward currents of NCX (recorded by whole-cell patch-clamp analysis) were reduced during 10-min hypoxia, and F(2) further inhibited the outward currents of NCX during 10-min hypoxia. All these data of SERCA2a and NCX might be responsible for the changes in calcium transients during hypoxia., Conclusion: Our data suggest that F(2) reduced changes in calcium transients that caused by hypoxia/ischemia, which was regarded to be a protective role in calcium homeostasis of ventricular myocytes, probably via changing the function of SERCA2a., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: To investigate the effect of desiccation on secretion of inflammatory cytokines in corneal epithelial cells and in the rat desiccation model., Methods: A human corneal epithelial cell line (CEPI) was grown in keratinocyte growth medium 2 (KGM2) to approximately 80% confluence. The medium was aspirated and dishes were left for 0 to 30 min with the cover left open to dry the cells (short-term desiccation). After desiccation, KGM2 was added to the dishes and collected from the dishes 15 min later to measure the concentrations of cytokines in the medium by sandwich enzyme immunoassay (ELISA). Viability of the cells was estimated with alamer blue. To study the effect of long-term desiccation, cultivated cells on transwells were used. After dessiccation for up to 8 h, the viability of the cells and levels of cytokines in the culture medium were examined. The expression of cytokines in the cornea of the dry eye model rat was measured by real-time PCR., Results: Short-term dessication of CEPI cells significantly increased the interleukin (IL)-6 level and slightly increased the tumor necrosis factor (TNF)-α level. Anti-IL-6 antibody partially suppressed cell death caused by desiccation. Upon long-term desiccation, IL-6 and IL-8 levels were increased. 
In the dry eye model rats, the IL-6 mRNA level in the cornea significantly increased, whereas TNF-α mRNA level slightly increased., Conclusions: Desiccation induced IL-6 expression in corneal epithelial cells, suggesting that IL-6 participates in desiccation-induced cell death.