Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Pi-complex structure of gaseous benzene-NO cations assayed by IR multiple photon dissociation spectroscopy

[C(6)H(6)NO](+) ions, in two isomeric forms involved as key intermediates in the aromatic nitrosation reaction, have been produced in the gas phase and analyzed by IR multiple photon dissociation (IRMPD) spectroscopy in the 800-2200 cm(-)(1) fingerprint wavenumber range, exploiting the high fluence and wide tunability of a free electron laser (FEL) source. The IRMPD spectra were compared with the IR absorption spectra calculated for the optimized structures of potential isomers, thus allowing structural information on the absorbing species. [C(6)H(6)NO](+) ions were obtained by two routes, taking advantage of the FEL coupling to two different ion traps. In the first one, an FT-ICR mass spectrometer, a sequence of ion-molecule reactions was allowed to occur, ultimately leading to an NO(+) transfer process to benzene. The so-formed ions displayed IRMPD features characteristic of a [benzene,NO](+) pi-complex structure, including a prominent band at 1963 cm(-)(1), within the range for the N-O bond stretching vibration of NO (1876 cm(-)(1)) and NO(+) (2344 cm(-)(1)). A quite distinct species is formed by electrospray ionization (ESI) of a methanol solution of nitrosobenzene. The ions transferred and stored in a Paul ion trap showed the IRMPD features of substituent protonated nitrosobenzene, the most stable among conceivable [C(6)H(6)NO](+) isomers according to computations. It is noteworthy that IRMPD is successful in allowing a discrimination between isomeric [C(6)H(6)NO](+) species, whereas high-energy collision-induced dissociation fails in this task. The [benzene,NO](+) pi-complex is characterized by IRMPD spectroscopy as an exemplary noncovalent ionic adduct between two important biomolecular moieties.     

Degree-independent Sobolev extension on locally uniform domains

We consider the problem of constructing extensions , where is the Sobolev space of functions with k derivatives in Lp and Ω⊂Rn is a domain. In the case of Lipschitz Ω, Calderón gave a family of extension operators depending on k, while Stein later produced a single (k-independent) operator. For the more general class of locally-uniform domains, which includes examples with highly non-rectifiable boundaries, a k-dependent family of operators was constructed by Jones. In this work we produce a k-independent operator for all spaces on a locally uniform domain Ω.     

Local Linear Convergence for Alternating and Averaged Nonconvex Projections

The idea of a finite collection of closed sets having “linearly regular intersection” at a point is crucial in variational analysis. This central theoretical condition also has striking algorithmic consequences: in the case of two sets, one of which satisfies a further regularity condition (convexity or smoothness, for example), we prove that von Neumann’s method of “alternating projections” converges locally to a point in the intersection, at a linear rate associated with a modulus of regularity. As a consequence, in the case of several arbitrary closed sets having linearly regular intersection at some point, the method of “averaged projections” converges locally at a linear rate to a point in the intersection. Inexact versions of both algorithms also converge linearly. Research of A.S. Lewis supported in part by National Science Foundation Grant DMS-0504032. Research of D.R. Luke supported in part by National Science Foundation Grant DMS-0712796.     

Imaging Mass Spectrometry: Hype or Hope?

Label-free imaging mass spectrometry provides a new look into different research areas. Will chemical mass microscopy on a biological system move from hype to hope?

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Surface neutralization/surface reionization (Ping–Pong) mass spectrometry

Experiments are reported that utilize surface neutralization of hyperthermal organic ions to obtain hyperthermal neutral species that are reionized on oxygen-activated rhenium surface and detected by mass spectrometry. A special Ping–Pong mass spectrometer was designed and coupled to a double-focusing mass spectrometer to allow energy and angle-resolved measurements. Neutralization of pyridine and benzene ions on gold, copper, and aluminum surfaces generated substantial ion currents into the collecting plate. The ion currents obtained on gold surfaces were found to depend on the recombination energy of the ion projectile. Hyperthermal neutrals coming off the first surface were found to mainly originate from hydrocarbon adsorbates.     

A Microreactor with Thousands of Subcompartments: Enzyme‐Loaded Liposomes within Polymer Capsules

Fully loaded : Noncovalent anchoring of liposomes into polymer multilayered films with cholesterol‐modified polymers allows the preparation of capsosomes—liposome‐compartmentalized polymer capsules (see picture). A quantitative enzymatic reaction confirmed the presence of active cargo within the capsosomes and was used to determine the number of subcompartments within this novel biomedical carrier system.

     

Development of a Gas-Tight Syringe Headspace GC-FID Method for the Detection of Ethanol,and a Description of the Legal and Practical Framework for Its Analysis,in Samples of English and Welsh Motorists’ Blood and Urine

Ethanol is the most commonly used recreational drug worldwide. This study describes the development and validation of a headspace gas chromatography flame ionisation detection (HS-GC-FID) method using dual columns and detectors for simultaneous separation and quantitation. The use of a dual-column, dual-detector HS-GC-FID to quantitate ethanol is a common analytical technique in forensic toxicology; however, most analytical systems utilise pressure-balance injection rather than a simplified gas-tight syringe, as per this technique. This study is the first to develop and validate a technique that meets the specifications of the United Kingdom’s requirements for road traffic toxicology testing using a Shimadzu GC-2014 gas-tight syringe. The calibration ranged from 10 to 400 mg/100 mL, with a target minimum linearity of r2 > 0.999, using tertiary butanol as the internal standard marker. The method has an expanded uncertainty at 99.73% confidence of 3.64% at 80 mg/100 mL, which is the blood alcohol limit for drink driving in England and Wales. In addition, at 200 mg%—the limit at which a custodial sentence may be imposed on the defendant—the expanded uncertainty was 1.95%. For both the 80 mg% and 200 mg% concentrations, no bias was present in the analytical method. This method displays sufficient separation for other alcohols, such as methanol, isopropanol, acetaldehyde, and acetone. The validation of this technique complies with the recommended laboratory guidelines set out by United Kingdom and Ireland Association of Forensic Toxicologists (UKIAFT), the recently issued Laboratory 51 guidelines by the United Kingdom Accreditation Service (UKAS), and the criteria set out by the California Code of Regulations (CCR), 17 CCR § 1220.1.     

Weak Noncovalent Interactions in Three Closely Related Adamantane-Linked 1,2,4-Triazole N-Mannich Bases: Insights from Energy Frameworks,Hirshfeld Surface Analysis,In Silico 11β-HSD1 Molecular Docking and ADMET Prediction

Structural analysis and docking studies of three adamantane-linked 1,2,4-triazole N-Mannich bases (1–3) are presented. Compounds 1, 2 and 3 crystallized in the monoclinic P2₁/c, P2₁ and P2₁/n space groups, respectively. Crystal packing of 1 was stabilized by intermolecular C-H⋯O interactions, whereas compounds 2 and 3 were stabilized through intermolecular C-H⋯N, C-H⋯S and C-H⋯π interactions. The energy frameworks for crystal structures of 1–3 were described. The substituent effect on the intermolecular interactions and their contributions were described on the basis of Hirshfeld surface analyses. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibition potential, pharmacokinetic and toxicity profiles of compounds 1–3 were determined using in silico techniques. Molecular docking of the compounds into the 11β-HSD1 active site showed comparable binding affinity scores (−7.50 to −8.92 kcal/mol) to the 11β-HSD1 co-crystallized ligand 4YQ (−8.48 kcal/mol, 11β-HSD1 IC₅₀ = 9.9 nM). The compounds interacted with key active site residues, namely Ser170 and Tyr183, via strong hydrogen bond interactions. The predicted pharmacokinetic and toxicity profiles of the compounds were assessed, and were found to exhibit excellent ADMET potential.