Biogrid--a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates

Culturing stem cells as free-floating aggregates in suspension facilitates large-scale production of cells in closed systems, for clinical use. To comply with GMP standards, the use of substances such as proteolytic enzymes should be avoided. Instead of enzymatic dissociation, the growing cell aggregates may be mechanically cut at passage, but available methods are not compatible with large-scale cell production and hence translation into the clinic becomes a severe bottle-neck. We have developed the Biogrid device, which consists of an array of micrometerscale knife edges, micro-fabricated in silicon, and a manifold in which the microgrid is placed across the central fluid channel. By connecting one side of the Biogrid to a syringe or a pump and the other side to the cell culture, the culture medium with suspended cell aggregates can be aspirated, forcing the aggregates through the microgrid, and ejected back to the cell culture container. Large aggregates are thereby dissociated into smaller fragments while small aggregates pass through the microgrid unaffected. As proof-of-concept, we demonstrate that the Biogrid device can be successfully used for repeated passage of human neural stem/progenitor cells cultured as so-called neurospheres, as well as for passage of suspension cultures of human embryonic stem cells. We also show that human neural stem/progenitor cells tolerate transient pressure changes far exceeding those that will occur in a fluidic system incorporating the Biogrid microgrids. Thus, by using the Biogrid device it is possible to mechanically passage large quantities of cells in suspension cultures in closed fluidic systems, without the use of proteolytic enzymes.     

Electrocatalytic oxygen evolution from water on a Mn(III-V) dimer model catalyst--a DFT perspective

A complete water oxidation and oxygen evolution reaction (OER) cycle is monitored by means of density functional theory (DFT). A biomimetic model catalyst, comprising a μ-OH bridged Mn(III-V) dimer truncated by acetylacetonate ligand analogs and hydroxides is employed. The reaction cycle is divided into four electrochemical hydrogen abstraction steps followed by a series of chemical steps. The former employ the tyrosine/tyrosyl redox couple acting as electron and proton sink, thus determining the reference potential. Stripping hydrogen from water leads to the formation of two highly unstable Mn(V)=O/Mn(IV)-O˙ moieties, which subsequently combine to form a μ-peroxy O-O bond. O(2) evolution results from subsequent consecutive replacement of the remaining Mn-O bonds by water. A Zener "spintronic" type mechanism for virtually barrierless O(2) evolution is found. The applicability of DFT is discussed and extended to include the rate-limiting steps in the OER. Rather than attempting to compute transition states where KS-DFT is unreliable, an upper bound for the activation barrier of the O-O bond formation step is estimated from the hessians of the relevant intermediates.     

Measurement of meropenem concentration in different human biological fluids by ultra-performance liquid chromatography-tandem mass spectrometry

Meropenem is a broad-spectrum antibiotic, often used for the empirical treatment of infections in critically ill patients with acute kidney injury. Meropenem has clinically insignificant protein binding and, as a carbapenem antibiotic, shows time-dependent bacterial killing, meaning that the unbound or free antibiotic concentration in blood should be maintained above the minimal inhibitory concentration of the pathogen for at least 40 % of the dosing interval. We developed and validated simple chromatographic methods by ultra-performance liquid chromatography-tandem mass spectrometry to measure plasma, filtrate-dialysate, and urine concentrations of meropenem. Chromatographic separation was achieved using an Acquity^® UPLC^® BEH^TM (2.1?×?100 mm id, 1.7 μm) reverse-phase C₁₈ column, with a water/acetonitrile linear gradient containing 0.1 % formic acid at a 0.4-mL/min flow rate. Meropenem and its internal standard (ertapenem) were detected by electrospray ionization mass spectrometry in positive ion multiple reaction monitoring mode. The limits of quantification were 0.27, 0.24, and 1.22 mg/L, and linearity was observed between 0.27–150, 0.24–150, and 1.22–2,000 mg/L for plasma, filtrate-dialysate, and urine samples, respectively. Coefficients of variation and relative biases were less than 13.5 and 8.0 % for all biological fluids. Recovery values were greater than 68.3 %. Evaluation of the matrix effect showed ion suppression for meropenem and ertapenem. No carry-over was observed. The validated methods are useful for both therapeutic drug monitoring and pharmacokinetic studies. It could be applied to daily clinical laboratory practice to measure the concentration of meropenem in plasma, filtrate-dialysate, and urine.

Noise sensitivity in continuum percolation

We prove that the Poisson Boolean model, also known as the Gilbert disc model, is noise sensitive at criticality. This is the first such result for a Continuum Percolation model, and the first which involves a percolation model with critical probability p _c ≠ 1/2. Our proof uses a version of the Benjamini-Kalai-Schramm Theorem for biased product measures. A quantitative version of this result was recently proved by Keller and Kindler. We give a simple deduction of the non-quantitative result from the unbiased version. We also develop a quite general method of approximating Continuum Percolation models by discrete models with p _c bounded away from zero; this method is based on an extremal result on non-uniform hypergraphs.     

Beamforming with a circular microphone array for localization of environmental noise sources

It is often enough to localize environmental sources of noise from different directions in a plane. This can be accomplished with a circular microphone array, which can be designed to have practically the same resolution over 360°. The microphones can be suspended in free space or they can be mounted on a solid cylinder. This investigation examines and compares two techniques based on such arrays, the classical delay-and-sum beamforming and an alternative method called circular harmonics beamforming. The latter is based on decomposing the sound field into a series of circular harmonics. The performance of the two signal processing techniques is examined using computer simulations, and the results are validated experimentally.     

Infrared laser stark shift spectroscopy in ammonia

The Stark effect in ammonia has been theoretically and experimentally analyzed using lead salt tunable diode laser absorption spectroscopy and CO₂ laser absorption spectroscopy of several absorption lines around 1050 cm^–1 applied to an all-optical sensor for measuring of electric field strength. Measurements of the Stark splitting effect of theaR(5,K) ammonia lines forK=1–5 as well as for the sR(3,K) lines forK=0–3 have been made at Doppler broadening pressures and for several different electric field strengths. Theoretical electric field dependent energy levels have been evaluated by diagonalization of a 6×6 energy matrix constructed using both electric field independent and dependent terms. From the theoretical analysis the resolution can be predicted and optimized both in the Doppler broadened and in the pressure broadened regimes. The predicted resolution is 0.5% at an electric field strength of 20 kV/cm. The theoretical calculations and the experimental data recorded with the tunable diode laser system were compared with independent measurements made with a CO₂ laser system. The agreement between experimentally recorded and theoretically calculated spectra is good which indicates that the theoretical model is satisfactory for our purposes. The contribution from the normally forbidden ssR(5, 3) ammonia line to the absorption at theP(12) CO₂ laser line in the 9 m band is briefly discussed.