Diagnostic cellular organization features extracted from autofluorescence images

Depth-resolved NADH autofluorescence images are shown to differentiate between normal and precancerous engineered tissues. An inverse power law behavior of the power spectral density (PSD) of these images is observed, indicating a self-affine organization of mitochondrial NADH at length scales 1-10 microm. Power exponents of the PSD functions vary significantly with tissue depth and precancerous state, giving insight into the morphological changes associated with precancerous lesions and providing substantial potential for noninvasive clinical diagnosis of squamous epithelial lesions and tumors.     

Three‐Phase Barker Arrays

A 3‐phase Barker array is a matrix of third roots of unity for which all out‐of‐phase aperiodic autocorrelations have magnitude 0 or 1. The only known truly two‐dimensional 3‐phase Barker arrays have size 2 × 2 or 3 × 3. We use a mixture of combinatorial arguments and algebraic number theory to establish severe restrictions on the size of a 3‐phase Barker array when at least one of its dimensions is divisible by 3. In particular, there exists a double‐exponentially growing arithmetic function T such that no 3‐phase Barker array of size with exists for all . For example, , , and . When both dimensions are divisible by 3, the existence problem is settled completely: if a 3‐phase Barker array of size exists, then .     

High-Field Asymmetric-Waveform Ion Mobility Spectrometry and Electron Detachment Dissociation of Isobaric Mixtures of Glycosaminoglycans

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is shown to be capable of resolving isomeric and isobaric glycosaminoglycan negative ions and to have great utility for the analysis of this class of molecules when combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and tandem mass spectrometry. Electron detachment dissociation (EDD) and other ion activation methods for tandem mass spectrometry can be used to determine the sites of labile sulfate modifications and for assigning the stereochemistry of hexuronic acid residues of glycosaminoglycans (GAGs). However, mixtures with overlapping mass-to-charge values present a challenge, as their precursor species cannot be resolved by a mass analyzer prior to ion activation. FAIMS is shown to resolve two types of mass-to-charge overlaps. A mixture of chondroitin sulfate A (CSA) oligomers with 4–10 saccharides units produces ions of a single mass-to-charge by electrospray ionization, as the charge state increases in direct proportion to the degree of polymerization for these sulfated carbohydrates. FAIMS is shown to resolve the overlapping charge. A more challenging type of mass-to-charge overlap occurs for mixtures of diastereomers. FAIMS is shown to separate two sets of epimeric GAG tetramers. For the epimer pairs, the complexity of the separation is reduced when the reducing end is alkylated, suggesting that anomers are also resolved by FAIMS. The resolved components were activated by EDD and the fragment ions were analyzed by FTICR-MS. The resulting tandem mass spectra were able to distinguish the two epimers from each other.

Biomolecular Imaging with a C60-SIMS/MALDI Dual Ion Source Hybrid Mass Spectrometer: Instrumentation,Matrix Enhancement,and Single Cell Analysis

We describe a hybrid MALDI/C₆₀-SIMS Q-TOF mass spectrometer and corresponding sample preparation protocols to image intact biomolecules and their fragments in mammalian spinal cord, individual invertebrate neurons, and cultured neuronal networks. A lateral spatial resolution of 10 μm was demonstrated, with further improvement feasible to 1 μm, sufficient to resolve cell outgrowth and interconnections in neuronal networks. The high mass resolution (>13,000 FWHM) and tandem mass spectrometry capability of this hybrid instrument enabled the confident identification of cellular metabolites. Sublimation of a suitable matrix, 2,5-dihydroxybenzoic acid, significantly enhanced the ion signal intensity for intact glycerophospholipid ions from mammalian nervous tissue, facilitating the acquisition of high-quality ion images for low-abundance biomolecules. These results illustrate that the combination of C₆₀-SIMS and MALDI mass spectrometry offers particular benefits for studies that require the imaging of intact biomolecules with high spatial and mass resolution, such as investigations of single cells, subcellular organelles, and communities of cells. Graphical Abstract

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The development and assessment of high‐throughput mass spectrometry‐based methods for the quantification of a nanoparticle drug delivery agent in cellular lysate

The safe use of lipid‐based drug delivery agents requires fast and sensitive qualitative and quantitative assessment of their cellular interactions. Many mass spectrometry (MS) based analytical platforms can achieve such task with varying capabilities. Therefore, four novel high‐throughput MS‐based quantitative methods were evaluated for the analysis of a small organic gene delivery agent: N,N‐bis(dimethylhexadecyl)‐1,3‐propane‐diammonium dibromide (G16‐3). Analysis utilized MS instruments that detect analytes using low‐resolution tandem MS (MS/MS) analysis (i.e. QTRAP or linear ion trap in this work) or high‐resolution MS analysis (i.e. time of flight (ToF) or Orbitrap). Our results indicate that the validated fast chromatography (FC)‐QTRAP‐MS/MS, FC‐ LTQ‐Orbitrap‐MS, desorption electrospray ionization‐collision‐induced dissociation (CID)‐MS/MS and matrix assisted laser desorption ionization‐ToF/ToF‐MS MS methods were superior in the area of method development and sample analysis time to a previously developed liquid chromatography (LC)‐CID‐MS/MS. To our knowledge, this is the first evaluation of the abilities of five MS‐based quantitative methods that target a single pharmaceutical analyte. Our findings indicate that, in comparison to conventional LC‐CID‐MS/MS, the new MS‐based methods resulted in a (1) substantial reduction in the analysis time, (2) reduction in the time required for method development and (3) production of either superior or comparable quantitative data. The four new high‐throughput MS methods, therefore, were faster, more efficient and less expensive than a conventional LC‐CID‐MS/MS for the quantification of the G16‐3 analyte within tissue culture. When applied to cellular lysate, no significant change in the concentration of G16‐3 gemini surfactant within PAM212 cells was observed between 5 and 53 h, suggesting the absence of any metabolism/excretion from PAM212 cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Naphthocrown‐Strapped Calix[4]pyrroles: Formation of Self‐Assembled Structures by Ion‐Pair Recognition

A new approach to the construction of self‐assembled structures is reported that is based on ion‐pair recognition. Towards this end, the calix[4]pyrrole naphthocrown‐4 hybrid structures 2 and 3 were prepared. These multitopic receptors contain recognition sites for both anions and cations. On the basis of solution‐phase ¹H NMR spectroscopic analysis and solid‐state single‐crystal X‐ray diffraction structural studies, it was established that receptors 2 and 3 are able to bind specific ion pairs with high selectivity via different binding modes. In the case of CsF and CsCl, the ion‐pair complexes formed from receptors 2 and 3 were found to self‐assemble to produce either linear supramolecular polymeric crystalline solids or nanotube‐like cyclic hexamers depending on the specific choice of ion pairs and crystallization solvents. Proton NMR studies provided evidence for solution‐phase self‐association in organic media.     

Batch versus Flow Photochemistry: A Revealing Comparison of Yield and Productivity

The use of flow photochemistry and its apparent superiority over batch has been reported by a number of groups in recent years. To rigorously determine whether flow does indeed have an advantage over batch, a broad range of synthetic photochemical transformations were optimized in both reactor modes and their yields and productivities compared. Surprisingly, yields were essentially identical in all comparative cases. Even more revealing was the observation that the productivity of flow reactors varied very little to that of their batch counterparts when the key reaction parameters were matched. Those with a single layer of fluorinated ethylene propylene (FEP) had an average productivity 20 % lower than that of batch, whereas three‐layer reactors were 20 % more productive. Finally, the utility of flow chemistry was demonstrated in the scale‐up of the ring‐opening reaction of a potentially explosive [1.1.1] propellane with butane‐2,3‐dione.     

Enzymatic Desymmetrising Redox Reactions for the Asymmetric Synthesis of Biaryl Atropisomers

Atropisomeric biaryls carrying ortho‐hydroxymethyl and formyl groups were made enantioselectively by desymmetrisation of dialdehyde or diol substrates. The oxidation of the symmetrical diol substrates was achieved using a variant of galactose oxidase (GOase), and the reduction of the dialdehydes using a panel of ketoreductases. Either M or P enantiomers of the products could be formed, with absolute configurations assigned by time‐dependent DFT calculations of circular dichroism spectra. The differing selectivities observed with different biaryl structures offer an insight into the detailed structure of the active site of the GOase enzyme.     

Magnetically Aligned Supramolecular Hydrogels

The magnetic‐field‐induced alignment of the fibrillar structures present in an aqueous solution of a dipeptide gelator, and the subsequent retention of this alignment upon transformation to a hydrogel upon the addition of CaCl₂ or upon a reduction in solution pH is reported. Utilising the switchable nature of the magnetic field coupled with the slow diffusion of CaCl₂, it is possible to precisely control the extent of anisotropy across a hydrogel, something that is generally very difficult to do using alternative methods. The approach is readily extended to other compounds that form viscous solutions at high pH. It is expected that this work will greatly expand the utility of such low‐molecular‐weight gelators (LMWG) in areas where alignment is key.