Fecal 1H-NMR Metabolomics: A Comparison of Sample Preparation Methods for NMR and Novel in Silico Baseline Correction.

Analysis of enteric microbiota function indirectly through the fecal metabolome has the potential to be an informative diagnostic tool. However, metabolomic analysis of feces is hampered by high concentrations of macromolecules such as proteins, fats, and fiber in samples. Three methods-ultrafiltration (UF), Bligh-Dyer (BD), and no extraction (samples added directly to buffer, vortexed, and centrifuged)-were tested on multiple rat (n = 10) and chicken (n = 8) fecal samples to ascertain whether the methods worked equally well across species and individuals. An in silico baseline correction method was evaluated to determine if an algorithm could produce spectra similar to those obtained via UF. For both rat and chicken feces, UF removed all macromolecules and produced no baseline distortion among samples. By contrast, the BD and no extraction methods did not remove all the macromolecules and produced baseline distortions. The application of in silico baseline correction produced spectra comparable to UF spectra. In the case of no extraction, more intense peaks were produced. This suggests that baseline correction may be a cost-effective method for metabolomic analyses of fecal samples and an alternative to UF. UF was the most versatile and efficient extraction method; however, BD and no extraction followed by baseline correction can produce comparable results.

Validation of isolated metabolites from drug metabolism studies as analytical standards by quantitative NMR.

In discovery and development, having a qualified metabolite standard is advantageous. Chemical synthesis of metabolite standards is often difficult and expensive. As an alternative, biological generation and isolation of metabolites in the nanomole range are readily feasible. However, without an accurately defined concentration, these isolates have limited utility as standards. There is a significant history of NMR as both a qualitative and a quantitative technique, and these concepts have been merged recently to provide both structural and quantitative information on biologically generated isolates from drug metabolism studies. Previous methodologies relied on either specialized equipment or the use of an internal standard to the isolate. We have developed a technique in which a mathematically generated signal can be inserted into a spectrum postacquisition and used as a quantitative reference: artificial signal insertion for calculation of concentration observed (aSICCO). This technique has several advantages over previous methodologies. Any region in the analyte spectra, free from interference, can be chosen for the reference signal. In addition, the magnitude of the inserted signal can be modified to appropriately match the intensity of the sample resonances. Because this is postacquisition quantification, no special equipment or pulse sequence is needed. Compared with quantitation via the addition of an internal standard (10 mM maleic acid), the signal insertion method produced similar results. For each method, precision and accuracy were within ± 5%, stability of signal response over 8 days was ± 5%, and the dynamic range was more than 3 orders of magnitude: 10 to 0.01 mM.

Methodology for Single Bee and Bee Brain 1H-NMR Metabolomics.

The feasibility of metabolomic 1H NMR spectroscopy is demonstrated for its potential to help unravel the complex factors that are impacting honeybee health and behavior. Targeted and non-targeted 1H NMR metabolic profiles of liquid and tissue samples of organisms could provide information on the pathology of infections and on environmentally induced stresses. This work reports on establishing extraction methods for NMR metabolic characterization of Apis mellifera, the European honeybee, describes the currently assignable aqueous metabolome, and gives examples of diverse samples (brain, head, body, whole bee) and biologically meaningful metabolic variation (drone, forager, day old, deformed wing virus). Both high-field (600 MHz) and low-field (80 MHz) methods are applicable, and 1H NMR can observe a useful subset of the metabolome of single bees using accessible NMR instrumentation (600 MHz, inverse room temperature probe) in order to avoid pooling several bees. Metabolite levels and changes can be measured by NMR in the bee brain, where dysregulation of metabolic processes has been implicated in colony collapse. For a targeted study, the ability to recover 10-hydroxy-2-decenoic acid in mandibular glands is shown, as well as markers of interest in the bee brain such as GABA (4-aminobutyrate), proline, and arginine. The findings here support the growing use of 1H NMR more broadly in bees, native pollinators, and insects.

Thermoresponsive copolymer nanofilms for controlling cell adhesion, growth, and detachment.

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.

Use of the 1-mm micro-probe for metabolic analysis on small volume biological samples.

Endogenous metabolites are promising diagnostic end-points in cancer research. Clinical application of high-resolution NMR spectroscopy is often limited by extremely low volumes of human specimens. In the present study, the use of the Bruker 1-mm high-resolution TXI micro-probe was evaluated in the elucidation of metabolic profiles for three different clinical applications with limited sample sizes (body fluids, isolated cells and tissue biopsies). Sample preparation and (1)H-NMR metabolite quantification protocols were optimized for following oncology-oriented applications: (i) to validate the absolute concentrations of citrate and spermine in human expressed prostatic specimens (EPS volumes 5 to 10 microl: prostate cancer application); (ii) to establish the metabolic profile of isolated human lymphocytes (total cell count 4 x 10(6): chronic myelogenous leukaemia application); (iii) to assess the metabolic composition of human head-and-neck cancers from mouse xenografts (biopsy weights 20 to 70 mg: anti-cancer treatment application). In this study, the use of the Bruker 1-mm micro-probe provides a convenient way to measure and quantify endogenous metabolic profiles of samples with a very low volume/weight/cell count.

Surface structural conformations of fibrinogen polypeptides for improved biocompatibility.

This work reports on how incorporation of silica nanocages into poly(urethane) copolymers (PU) affects conformational orientations of adsorbed fibrinogen and how different surfaces subsequently influenced HeLa cell attachment and proliferation. Incorporation of 2 wt% silica nanocages into poly(urethane) (PU4) substantially altered the surface topography of the films and some 50% of the surface was covered with the nanocages due to their preferential exposure. AFM studies revealed the deposition of a dense protein network on the soft polymeric domains of PU4 and much reduced fibrinogen adsorption on the hard nanocage domains. As on the bare SiO(2) control surface, fibrinogen molecules adsorbed on top of the hard nanocages mainly took the dominant trinodular structures in monomeric and dimeric forms. In addition, net positively charged long alpha chains were prone to being hidden beneath the D domains whilst gamma chains predominantly remained exposed. Dynamic interfacial adsorption as probed by spectroscopic ellipsometry revealed fast changes in interfacial conformation induced by electrostatic interactions between different segments of fibrinogen and the surface, consistent with the AFM imaging. On the PU surfaces without nanocage incorporation (PUA), however, adsorbed fibrinogen molecules formed beads-like chain networks, consistent with the structure featured on the soft PU4 domains, showing very different effects of surface chemical nature. Monoclonal antibodies specific to the alpha and gamma chains showed reduced alpha but increased gamma chain binding at the silicon oxide control and PU4 surfaces, whilst on the PUA, C18 and amine surfaces (organic surface controls) the opposite binding trend was detected with alpha chain binding dominant, showing different fibrinogen conformations. Cell attachment studies revealed differences in cell attachment and proliferation, consistent with the different polypeptide conformations on the two types of surfaces, showing a strong preference to the extent of exposure of gamma chains.

Sex pheromone of the longtailed mealybug: a new class of monoterpene structure.

The sex pheromone of the longtailed mealybug, identified as 2-(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate, represents the first example of a new monoterpenoid skeleton. A [2,3]-sigmatropic rearrangement was used in a key step during construction of the sterically congested tetraalkylcylopentene framework.