ECM-transmitted shear stress induces apoptotic cell extrusion in early breast gland development.

Epithelial cells of human breast glands are exposed to various mechanical ECM stresses that regulate tissue development and homeostasis. Mechanoadaptation of breast gland tissue to ECM-transmitted shear stress remained poorly investigated due to the lack of valid experimental approaches. Therefore, we created a magnetic shear strain device that enabled, for the first time, to analyze the instant shear strain response of human breast gland cells. MCF10A-derived breast acini with basement membranes (BM) of defined maturation state and basoapical polarization were used to resemble breast gland morphogenesis in vitro. The novel biophysical tool was used to apply cyclic shear strain with defined amplitudes (≤15%, 0.2 Hz) over 22 h on living spheroids embedded in an ultrasoft matrix (<60 Pa). We demonstrated that breast spheroids gain resistance to shear strain, which increased with BM maturation and basoapical polarization. Most intriguingly, poorly developed spheroids were prone to cyclic strain-induced extrusion of apoptotic cells from the spheroid body. In contrast, matured spheroids were insensitive to this mechanoresponse-indicating changing mechanosensing or mechanotransduction mechanisms during breast tissue morphogenesis. Together, we introduced a versatile tool to study cyclic shear stress responses of 3D cell culture models. It can be used to strain, in principle, all kinds of cell clusters, even those that grow only in ultrasoft hydrogels. We believe that this approach opens new doors to gain new insights into dynamic shear strain-induced mechanobiological regulation circuits between cells and their ECM.

Optimized Continuous Application of Hyperpolarized Xenon to Liquids.

In recent years, NMR with hyperpolarized (HP) xenon inside functionalized host structures (e.g., cryptophanes) have become a potential candidate for the direct observation of metabolic processes (i.e., molecular imaging). A critical issue for real applications is the dissolution of the HP-gas in the liquid which contains the host. In this work, we present recent developments for an improved and controlled dissolution of HP-Xe in liquids using hollow fiber membranes and different compressor systems. The designed apparatus consists of a compressor and a membrane unit. The compressor provides HP-129Xe continuously at small adjustable pressures and in a polarization-preserving way. The membrane unit enables a molecular solution of the HP-gas in aqueous liquids, avoiding the formation of bubbles or even foams. Two different types of compressors were tested in terms of function and useful materials. Special emphasis was put on a systematic reduction of transfer losses in the gas and liquid phase. In order to optimize the system parameters, several physical models were developed to describe the transport and the losses of nuclear polarization. Finally, the successful implementation was demonstrated in several experiments. HP-Xe was dissolved in an aqueous cryptophane-A-(OCH2COOH)6 solution, and stable Xe signals could be measured over 35 min, only limited by the size of the gas reservoir. Such long and stable experimental conditions enabled the study of chemical exchange of xenon between cryptophane and water environments even for a time-consuming 2D NMR experiment. The good signal stability over the measurement time allowed an exact determination of the residence time of the Xe atom inside the cryptophane, resulting in an average residence time of 44.5 ± 2.7 ms.

How to Reach a Thousand-Second in-Plane Polarization Lifetime with 0.97-GeV/c Deuterons in a Storage Ring.

We observe a deuteron beam polarization lifetime near 1000 s in the horizontal plane of a magnetic storage ring (COSY). This long spin coherence time is maintained through a combination of beam bunching, electron cooling, sextupole field corrections, and the suppression of collective effects through beam current limits. This record lifetime is required for a storage ring search for an intrinsic electric dipole moment on the deuteron at a statistical sensitivity level approaching 10^{-29} e cm.

HP-Xe to go: Storage and transportation of hyperpolarized (129)Xenon.

Recently the spin-lattice relaxation time T1 of hyperpolarized (HP)-(129)Xe was significantly improved by using uncoated and Rb-free storage vessels of GE180 glass. For these cells, a simple procedure was established to obtain reproducible wall relaxation times of about 18 h. Then the limiting relaxation mechanism in pure Xe is due to the coupling between the nuclear spins and the angular momentum of the Xe-Xe van-der-Waals-molecules. This mechanism can be significantly reduced by using different buffer gases of which CO2 was discovered to be the most efficient so far. From these values, it was estimated that for a 1:1 mixture of HP-Xe with CO2 a longitudinal relaxation time of about 7 h can be expected, sufficient to transport HP-Xe from a production to a remote application site. This prediction was verified for such a mixture at a total pressure of about 1 bar in a 10 cm glass cell showing a storage time of T1≈9 h (for T1(wall)=(34±9) h) which was transported inside a magnetic box over a distance of about 200 km by car.

New Method for a Continuous Determination of the Spin Tune in Storage Rings and Implications for Precision Experiments.

A new method to determine the spin tune is described and tested. In an ideal planar magnetic ring, the spin tune-defined as the number of spin precessions per turn-is given by ν(s)=γG (γ is the Lorentz factor, G the gyromagnetic anomaly). At 970 MeV/c, the deuteron spins coherently precess at a frequency of ≈120 kHz in the Cooler Synchrotron COSY. The spin tune is deduced from the up-down asymmetry of deuteron-carbon scattering. In a time interval of 2.6 s, the spin tune was determined with a precision of the order 10^{-8}, and to 1×10^{-10} for a continuous 100 s accelerator cycle. This renders the presented method a new precision tool for accelerator physics; controlling the spin motion of particles to high precision is mandatory, in particular, for the measurement of electric dipole moments of charged particles in a storage ring.

Physical degradation of proteins in well-defined fluid flows studied within a four-roll apparatus.

In most applications of biotechnology and downstream processing proteins are exposed to fluid stresses in various flow configurations which often lead to the formation of unwanted protein aggregates. In this paper we present physical degradation experiments for proteins under well-defined flow conditions in a four-roll apparatus. The flow field was characterized numerically by computational fluid dynamics (CFD) and experimentally by particle image velocimetry (PIV). The local shear strain rate as well as the local shear and elongation rate was used to characterize the hydrodynamic stress environment acting on the proteins. Lysozyme was used as a model protein and subjected to well-defined fluid stresses in high and low stress environment. By using in situ turbidity measurements during stressing the aggregate formation was monitored directly in the fluid flow. An increase in absorbance at 350 nm was attributed to a higher content of visible particles (>1 µm). In addition to lysozyme, the formation of aggregates was confirmed for two larger proteins (bovine serum albumin and alcohol dehydrogenase). Thus, the presented experimental setup is a helpful tool to monitor flow-induced protein aggregation with high reproducibility. For instance, screening experiments for formulation development of biopharmaceuticals for fill and finish operations can be performed in the lab-scale in a short time-period if the stress distributions in the application are transferred and applied in the four-roll mill.

Influence of the first amplifier stage in MEA systems on extracellular signal shapes.

Recording of extracellular signals with planar metal microelectrodes (ME) has already been presented more than 30 years ago. To date, microelectrode array (MEA) systems are able to measure extracellular signals at about 64 sites, simultaneously. This enables monitoring of electrical activity of many cells in a large area. The extracellular recording technique has become a widely used method for neurological, toxicological or pharmacological studies. It already proved its potential to supplement the classical methods in electrophysiology. The interpretation of the recorded signal shapes in order to extract electrophysiological meaningful data--however--is still under discussion. In this article, we analyse the preamplifier circuit for extracellular recording of cardiac myocyte signals. We use a circuit model for the cell-electrode contact including the first amplification stage. In test experiments, we observe different signal shapes, when different shunt resistors are introduced at the input of the preamplifier. According to the frequency spectra of the recordings, we evaluate the transfer function between the source signal and the readout signal. As a result of our studies, an optimum readout electronics for originally, preserved extracellular signal shapes is proposed. Our amplifier design will be most valuable, if the use of small microelectrodes with high input impedances for in vitro as well as for in vivo experiments is desired.

Reproducibility and reliability of fetal cardiac time intervals using magnetocardiography.

We investigated several factors which may affect the accuracy of fetal cardiac time intervals (CTI) determined in magnetocardiographic (MCG) recordings: observer differences, the number of available recording sites and the type of sensor used in acquisition. In 253 fetal MCG recordings, acquired using different biomagnetometer devices between the 15th and 42nd weeks of gestation, P-wave, QRS complex and T-wave onsets and ends were identified in signal averaged data sets independently by different observers. Using a defined procedure for setting signal events, interobserver reliability was high. Increasing the number of registration sites led to more accurate identification of the events. The differences in wave morphology between magnetometer and gradiometer configurations led to deviations in timing whereas the differences between low and high temperature devices seemed to be primarily due to noise. Signal-to-noise ratio played an important overall role in the accurate determination of CTI and changes in signal amplitude associated with fetal maturation may largely explain the effects of gestational age on reproducibility. As fetal CTI may be of value in the identification of pathologies such as intrauterine growth retardation or fetal cardiac hypertrophy, their reliable estimation will be enhanced by strategies which take these factors into account.

Interactions of liposome-incorporated amphotericin B with kidney epithelial cell cultures.

The polyene antibiotic amphotericin B (AmB) is profoundly cytotoxic to both fungal cells and mammalian cells. We have previously shown that the incorporation of AmB into phospholipid vesicles can markedly reduce the toxicity of the drug for mammalian cells (erythrocytes) without changing its antifungal potency [Mol. Pharmacol. 31:1-11 (1987)]. Because the primary site of in vivo toxicity of AmB is the kidney, here we investigate the effects of free AmB and liposomal AmB (L-AmB) on LLCPK1 cells, a porcine kidney cell line with many characteristics typical of proximal tubule cells. Acute exposure (2 hr) to free AmB inhibits protein synthesis and causes cell detachment and protein loss in LLCPK1 cells, with an IC50 of about 30 micrograms/ml. By contrast, certain formulations of L-AmB have little effect on protein synthesis/protein loss at concentrations of up to 2 mg/ml. The action of liposomes in protecting against acute AmB toxicity extends to effects on sugar transport and on cellular morphology in differentiated cultured kidney cells. Thus, the IC50 for inhibition of sodium-stimulated glucose transport by free AmB is 1.5 micrograms/ml whereas concentrations of L-AmB up to 1 mg/ml do not inhibit this process. However, chronic exposure of cells to L-AmB results in profound toxic effects as manifested by changes in cellular transport functions and cell morphology. Our results suggest that extended periods of proximity between cells and liposomes permit the transfer to toxic amounts of AmB. This may be of importance to the therapeutic use of AmB, for which protracted courses of drug administration are common.

In vivo behavior of polymerized lipid vesicles.

We have investigated the blood clearance kinetics and tissue distribution of large multilamellar liposomes (MLVs) and small unilamellar liposomes (SUVs) composed of the photopolymerizable lipid bis[12-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine. Polymerized MLVs or SUVs are cleared from the bloodstream more rapidly than nonpolymerized vesicles with similar size profiles. Both polymerized and nonpolymerized vesicles primarily accumulate in liver and spleen. However, the polymerized vesicles display an enhanced biostability as revealed by an elevated level of radioactive lipid marker remaining in the tissues.

Membrane-to-membrane transfer of lipophilic drugs used against cancer or infectious disease.

Use of liposomal drug delivery systems can enhance the therapeutic potential of membrane active anti-cancer and anti-infectious drugs. Thus, the therapeutic index of the important antifungal agent amphotericin B is markedly improved via incorporation of the drug into liposomes. The mechanistic basis of this effect seems to be an increase in the selectivity of the drug at the cellular level. Thus, free amphotericin B can readily partition into both fungal and mammalian membranes and can cause toxicity to both types of cells, giving rise to the notorious in vivo toxicity of this drug. By contrast, when amphotericin B is formulated in certain types of liposomes, the drug still readily partitions into fungal membranes but can no longer partition into animal cell membranes, thus markedly reducing its toxicity. Liposomes can also be used to reduce the toxicity of membrane-active antitumor drugs. Thus, the peptide ionophore valinomycin is far less toxic to animals when presented in liposomal form. Nonetheless, the drug retains useful antitumor activity in this form. The underlying basis of the enhanced therapeutic index of liposomal valinomycin is unknown at this time but is being explored. The development of membrane-active anti-tumor drugs, in conjunction with liposomal delivery systems, could be an important new approach in cancer chemotherapy. While no anticancer drug is likely to be free of toxic side effects, the toxicities engendered by membrane-active antitumor drugs are likely to affect a different spectrum of tissues and organs than those caused by "conventional" antitumor drugs. Thus membrane-active drugs could complement existing drugs and provide a valuable adjunct to therapy.

Polymerized phospholipid vesicles containing amphotericin B: evaluation of toxic and antifungal activities in vitro.

We have prepared lipid vesicles (liposomes) composed of polymerized bis[12-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine (DPL) which contain the antifungal polyene antibiotic amphotericin B (AMB). It was necessary to devise a novel method for incorporating AMB into the liposomes subsequent to polymerization. The polymer liposome AMB was as effective as AMB in "conventional" liposomes in terms of inhibiting fungal growth in vitro. However, in contrast to "conventional" liposomes, the polymerized DPL vesicles did not protect mammalian cells against AMB induced toxicity.

Preparation of gelatin nanocapsules and their pharmaceutical characterization.

A production process is described that yields nanocapsules instead of nanoparticles. The process allows the encapsulation of lipophilic drugs such as triamcinolone acetonide with an O/W emulsion system. The capsular structure of the products was confirmed by transmission and scanning electron microscopy. The nanocapsules displayed a mean size of 141 ± 47 nm to 523 ± 340 nm (mean ± S.D.) and a drug content of 7 % to 15.4 % w/w. Drug release experiments showed that the encapsulated triamcinolone acetonide was released more slowly than micronized drug crystals. A more pronounced retarded release could be achieved by raising the pH during the hardening reaction or by encapsulating cholesterol into the nanocapsules.