Multivesicular droplets: a cell model system to study compartmentalised biochemical reactions.

Multivesicular vesicles (MVVs) are artificial liposomal structures widely used as a platform to study the compartmentalisation of cells and as a scaffold for artificial cell/protocell models. Current preparation techniques for MVVs, however, offer poor control on the size, lamellarity, and loading of inner lipid vesicles. Here, we introduce a microfluidic device for the production of multivesicular droplets (MVDs): a novel model system combining the ease of use and control of droplet microfluidics with the biological relevance of MVVs. We use a perfluorinated carrier phase with a biocompatible surfactant to generate monodisperse droplets of an aqueous giant unilamellar lipid vesicle suspension. The successful on-chip formation and stability of MVDs is verified through high-speed microscopy. For bright field or fluorescence microscopy inspection, the MVDs are trapped in an array where the integrity of both lipid vesicles and droplets is preserved for up to 15 minutes. Finally, we show a two-step enzymatic reaction that takes place across the lipid vesicle membranes; the second reaction step occurs in the vesicle's interior, where the enzyme is encapsulated, while both the substrate and fluorescent product permeate across the membrane. Our approach opens the possibility to mimic artificial organelles with optimised reaction parameters (pH, ions, etc.) in each compartment.

Characterisation of anticancer peptides at the single-cell level.

The development of efficacious anticancer therapeutics is difficult due to the heterogeneity of the cellular response to chemotherapy. Anticancer peptides (ACPs) are promising drug candidates that have been shown to be active against a range of cancer cells. However, few ACP studies focus on tumour single-cell heterogeneities. In order to address this need, we developed a microfluidic device and an imaging procedure that enable the capture, monitoring, and analysis of several hundred single cells for the study of drug response. MCF-7 human breast adenocarcinoma cells were captured in hydrodynamic traps and isolated in individual microchambers of less than 100 pL volume. With pneumatic valves, different sets of microchambers were actuated to expose the cells to various drugs. Here, the effect of three membranolytic ACPs - melittin, aurein 1.2 and aurein 2.2 - was investigated by monitoring the efflux of calcein from single MCF-7 cells. The loss of membrane integrity was observed with two different strategies that allow either focusing on one cell for mechanistic studies or parallel analysis of hundreds of individual cells. In general, the device is applicable to the analysis of the effect of various drugs on a large number of different cell types. The platform will enable us in the future to determine the origin of heterogeneous responses on pharmacological substances like ACPs within cell populations by combining it with other on-chip analytical methods.

Single cells in confined volumes: microchambers and microdroplets.

Microfluidic devices capable of manipulating and guiding small fluid volumes open new methodical approaches in the fields of biology, pharmacy, and medicine. They have already proven their extraordinary value for cell analysis. The emergence of microfluidic platforms has paved the way to novel analytical strategies for the positioning, treatment and observation of living cells, for the creation of chemically defined liquid environments, and for tailoring biomechanical or physical conditions in small volumes. In this article, we particularly focus on two complementary approaches: (i) the isolation of cells in small chambers defined by microchannels and integrated valves and (ii) the encapsulation of cells in microdroplets. We review the advantages and limitations of both approaches and discuss their potential for single-cell analysis and related fields. Our intention is also to give a recommendation on which platform is most appropriate for a new question, i.e., a guideline to choose the most suitable platform.

Chemical computing with reaction-diffusion processes.

Chemical reactions are responsible for information processing in living organisms. It is believed that the basic features of biological computing activity are reflected by a reaction-diffusion medium. We illustrate the ideas of chemical information processing considering the Belousov-Zhabotinsky (BZ) reaction and its photosensitive variant. The computational universality of information processing is demonstrated. For different methods of information coding constructions of the simplest signal processing devices are described. The function performed by a particular device is determined by the geometrical structure of oscillatory (or of excitable) and non-excitable regions of the medium. In a living organism, the brain is created as a self-grown structure of interacting nonlinear elements and reaches its functionality as the result of learning. We discuss whether such a strategy can be adopted for generation of chemical information processing devices. Recent studies have shown that lipid-covered droplets containing solution of reagents of BZ reaction can be transported by a flowing oil. Therefore, structures of droplets can be spontaneously formed at specific non-equilibrium conditions, for example forced by flows in a microfluidic reactor. We describe how to introduce information to a droplet structure, track the information flow inside it and optimize medium evolution to achieve the maximum reliability. Applications of droplet structures for classification tasks are discussed.

Controllable electrofusion of lipid vesicles: initiation and analysis of reactions within biomimetic containers.

We present a microfluidic device that is able to trap multiple giant unilamellar vesicles (GUVs) and initiate electrofusion via integrated microelectrodes. PDMS posts were designed to trap and isolate two or more vesicles. Electrodes patterned onto the glass surface of the microchannels are able to apply a short, high voltage pulse across the traps for controllable electrofusion of the GUVs. The entire array of traps and electrodes are designed such that an average of 60 individual fusion experiments can be performed on-chip. An assay based on Förster resonance energy transfer (FRET) is performed to show successful lipid mixing. Not only can the device be used to record the dynamics of lipid membrane fusion, but it can be used for reaction monitoring by fusing GUVs containing reactants. We demonstrate this by fusing vesicles encapsulating femtolitre volumes of cobalt chloride or EDTA and monitoring the amount of the complexation product over time.

Implementing enzyme-linked immunosorbent assays on a microfluidic chip to quantify intracellular molecules in single cells.

Cell-to-cell differences play a key role in the ability of cell populations to adapt and evolve, and they are considered to impact the development of several diseases. Recent advances in microsystem technology provide promising solutions for single-cell studies. However, the quantitative chemical analysis of single-cell lysates remains difficult. Here, we combine a microfluidic device with the analytical strength of enzyme-linked immunosorbent assays (ELISA) for single-cell studies to reliably identify intracellular proteins, secondary messengers, or metabolites. The microfluidic device allows parallel single-cell trapping and isolation in 625-pL microchambers, repeated treatment and washing steps, subsequent lysis and analysis by ELISA. Using a sandwich ELISA, we quantitatively determined the concentration of the enzyme GAPDH in single U937 cells and HEK 293 cells, and found amounts within a range of a few (1-4) attomol per cell. Furthermore, a competitive ELISA is performed to determine the concentration of the secondary messenger cyclic adenosine monophosphate (cAMP) in MLT cells, in response to the hormone lutropin. We found the half maximal effective concentration (EC50) of lutropin to have an average value of 2.51 ± 0.44 ng/mL. Surprisingly, there were large cell-to-cell variations for all supplied lutropin concentrations, ranging from 36 to 536 attomol cAMP for nonstimulated cells and from 80 to 1040 attomol cAMP for a concentration around the EC50 (3 ng/mL). Because of the high sensitivity and specificity of ELISA and the large number of antibodies available, we believe that our device provides a new, powerful means for single-cell proteomics and metabolomics.

Microfluidic trapping of giant unilamellar vesicles to study transport through a membrane pore.

We present a microfluidic platform able to trap single GUVs in parallel. GUVs are used as model membranes across many fields of biophysics including lipid rafts, membrane fusion, and nanotubes. While their creation is relatively facile, handling and addressing single vesicles remains challenging. The PDMS microchip used herein contains 60 chambers, each with posts able to passively capture single GUVs without compromising their integrity. The design allows for circular valves to be lowered from the channel ceiling to isolate the vesicles from rest of the channel network. GUVs containing calcein were trapped and by rapidly opening the valves, the membrane pore protein α-hemolysin (αHL) was introduced to the membrane. Confocal microscopy revealed the kinetics of the small molecule efflux for different protein concentrations. This microfluidic approach greatly improves the number of experiments possible and can be applied to a wide range of biophysical applications.

Monitoring induced gene expression of single cells in a multilayer microchip.

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.

Pharmacokinetics of a novel liquid controlled release codeine formulation.

Codeine is an important opioid anti-tussive agent whose short half-life (2.9 ± 0.7 h) requires that it be administered at 4-h intervals when formulated as a simple aqueous solution. Liquid controlled release codeine formulations such as an older Codipertussin(®) formulation, which contained codeine bound to an ion exchange resin and coated with a retardant polymer, achieved an equivalent bioavailability when administered every 12 h. An accompanying paper described the development and in vitro characterization of a novel Codipertussin(®) formulation containing a non-coated codeine:ion exchange resin (Amberlite IR 69 F) complex. In this study, the bioavailability of codeine from this new liquid controlled release formulation was investigated in an open label, single center, randomized, steady-state, cross-over study in healthy male volunteers. Participants received either 69.7 mg codeine as the controlled release liquid form every 12 h or 23.2 mg codeine in solution every 4 h. Controlled release from the suspension of beads protracted the apparent mean half life of codeine from 3.2 h to 8.2 h, while the mean AUC(0-12 h) was unchanged. In vivo codeine release profiles were further derived by the numerical deconvolution method, using the data from the drug solution as weighting function for the body system. Comparison of the data obtained with the in vitro release data presented in our earlier work showed an acceptable in vitro-in vivo correlation, which was described as in vitro-in vivo relationship, indicating the power of the in vitro method to predict in vivo pharmacokinetic behavior.

Bilayer microfluidic chip for diffusion-controlled activation of yeast species.

A bilayer microfluidic chip is used, in which multiple laminar streams are generated to define local microenvironments. The bilayer architecture of the microchip separates cell handling and positioning from cell activation by soluble chemicals. Cell activation is diffusion controlled through a porous membrane. By employing time-lapse fluorescence microscopy, gene expression of the enhanced green fluorescent protein (eGFP) in Saccharomyces cerevisiae is studied under various conditions. We demonstrate that the yeast cells remain viable in the microchip for at least 17 h, and that gene expression can be initiated by the supply of the inducer galactose at a spatial precision of a few micrometers.

Immune complexes of auto-antibodies against A beta 1-42 peptides patrol cerebrospinal fluid of non-Alzheimer's patients.

The diagnostic potential of large A beta-peptide binding particles (LAPs) in the cerebrospinal fluid (CSF) of Alzheimer's dementia (AD) patients and non-AD controls (nAD) was evaluated. LAPs were detected by confocal spectroscopy in both groups with high inter-individual variation in number. Molecular imaging by confocal microscopy revealed that LAPs are heterogeneous superaggregates that could be subdivided morphologically into four main types (LAP 1-4). LAP-4 type, resembling a 'large chain of pearls', was detected in 42.1% of all nAD controls but it was virtually absent in AD patients. LAP-4 type could be selectively removed by protein A beads, a clear indication that it contained immunoglobulins in addition to beta-amyloid peptides (A beta 1-42). We observed a close correlation between LAPs and immunoglobulin G (IgG) concentration in CSF in controls but not in AD patients. Double labeling of LAPs with anti-A beta and anti-IgG antibodies confirmed that LAP-4 type consisted of A beta and IgG aggregates. Our results assign a central role to the immune system in regulating A beta1-42 homeostasis by clustering this peptide in immunocomplexes.

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Penetration of piperacillin and tazobactam into inflamed soft tissue of patients with diabetic foot infection.

We investigated the pharmacokinetics of piperacillin and tazobactam in the extracellular space fluid of inflamed soft tissues of six patients with diabetic foot infection using in vivo microdialysis and found similar penetration for piperacillin but not for tazobactam into inflamed and noninflamed soft tissue.

Characterization of the photoconversion on reaction of the fluorescent protein Kaede on the single-molecule level.

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with lambda = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (lambda = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at lambda = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(-1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.

Biphasic insulin aspart compared to biphasic human insulin reduces postprandial hyperlipidemia in patients with Type 2 diabetes.

BACKGROUND: Premixed insulin analogues reduce postprandial hyperglycemia in patients with Type 2 diabetes in comparison to premixed regular insulin. Insulin also plays an important role in the regulation of postprandial lipid metabolism. It is known that increased levels of postprandial insulin reduce postprandial hyperlipemia but, on the other hand, no information exists with regard to the possible effect of insulin analogues in comparison to human insulin. MATERIALS AND METHODS: 12 subjects (3 men; age 59 +/- 5 years; BMI 30.5 +/- 5.9 kg/m2, duration of diabetes 9 +/- 1 years, HbA1c 8.33 +/- 1.1 %) already on therapy with premixed insulin were treated either with biphasic human insulin (BHI30) or with biphasic insulin aspart (BIAsp30) (1.3 IU fast acting insulin/12 g KH) in the setting of a standardized test meal. Serum levels of glucose, insulin, C-peptide and triglycerides as well as retinylpalmitate in plasma and chylomicron remnants were determined before and up to 8 hours after the meal. RESULTS: As was to be expected, therapy with BIAsp30 reduced the maximum increase of postprandial glucose from 7.10 +/- 2.00 mmol/l to 5.27 +/- 1.83 mmo/l (p = 0.007) compared to BHI30 insulin. In the same way, the maximum increase of triglycerides (from 2.33 +/- 1.03 to 1.65 +/- 0.69 mmol/l, p = 0.014) was reduced. The AUC 0 - 8 for triglycerides was not significantly influenced (34.20 +/- 19.86 vs. 31.46 +/- 16.21 mmol x 8 h/l) but the incremental area over baseline (AOB 0 - 8) was significantly reduced from 8.02 +/- 4.35 to 6.12 +/- 3.94 mmol x 8 h/l (p = 0.024). CONCLUSIONS: Compared to conventional human premixed insulin the prandial therapy with biphasic insulin aspart results not only in an improvement of glucose tolerance but also in a significant reduction of postprandial hyperlipemia.

Penetration of fosfomycin into inflammatory lesions in patients with cellulitis or diabetic foot syndrome.

We investigated the distribution of the broad-spectrum antibiotic fosfomycin in infected soft tissue of patients with uncomplicated cellulitis of the lower extremities or diabetic foot infection using in vivo microdialysis. Our findings suggest that fosfomycin exhibits good and similar penetration into the fluid in the interstitial space in inflamed and noninflamed soft tissue in patients.

Artificial chemistries--a review.

This article reviews the growing body of scientific work in artificial chemistry. First, common motivations and fundamental concepts are introduced. Second, current research activities are discussed along three application dimensions: modeling, information processing, and optimization. Finally, common phenomena among the different systems are summarized. It is argued here that artificial chemistries are "the right stuff" for the study of prebiotic and biochemical evolution, and they provide a productive framework for questions regarding the origin and evolution of organizations in general. Furthermore, artificial chemistries have a broad application range of practical problems, as shown in this review.

Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states.

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state.

Effects of a high-fat meal on resistance vessel reactivity and on indicators of oxidative stress in healthy volunteers.

High fat meals postprandially impair macrovascular endothelial function and a link to increased oxidative stress is suggested. Few information, on the other hand, exists on the effect of postprandial hyperlipidaemia on resistance vessel function. Under normal circumstances this vascular bed regulates tissue perfusion and, by controlling flow, impacts on macrovascular nitric oxide formation. The impact of a high fat meal (1200 kcal, 90 g fat, 46 g protein and 47 g carbohydrates) on postprandial resistance vessel reactivity and on indicators of oxidative stress was studied in 11 healthy subjects by venous-occlusion plethysmography using another six subjects as time control group. Ingestion of the test meal resulted in a pronounced increase of serum triglycerides from 1.05 +/- 0.61 mmol l(-1) in the fasting state to peak postprandial values of 1.94 +/- 0.41 mmol l(-1) (P < 0.001) reached after 4 h and a return to baseline after 8 h. Fasting peak reactive hyperaemia (RH) was 19.6 +/- 2.4 ml min(-1) (100 ml)(-1). Two hours after ingestion of the test meal peak RH was transiently reduced to 16.8 +/- 2.2 ml min(-1) (100 ml)(-1) (P < 0.05). No alteration of resting forearm perfusion was observed. The time course of peak RH suggested a potential biphasic effect of the test meal with an early impairment and a late increase of RH. Ingestion of a lipid rich test meal did not exert any influence on either total plasma antioxidant capacity given in trolox equivalents (513 +/- 26 micromol l(-1) at baseline) or on plasma peroxides measured as H2O2 equivalents (469 +/- 117 micromol l(-1)). Our results suggest that ingestion of a meal containing 90 g of fat results in a transient impairment of reactive hyperaemia in healthy subjects but these vascular alterations are not accompanied by signs of systemically increased oxidative stress.

Accessing molecular dynamics in cells by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffraction-limited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.