Which Specialized Metabolites Does the Native Subantarctic Gastropod Notodiscus hookeri Extract from the Consumption of the Lichens Usnea taylorii and Pseudocyphellaria crocata?

Alice Gadea, Pierre Le Pogam, Grichka Biver, Joël Boustie, Anne-Cécile Le Lamer, Le Dévéhat Françoise, Maryvonne Charrier

Notodiscus hookeri is the only representative of terrestrial gastropods on Possession Island and exclusively feeds on lichens. The known toxicity of various lichen metabolites to plant-eating invertebrates led us to propose that N. hookeri evolved means to protect itself from their adverse effects. To validate this assumption, the current study focused on the consumption of two lichen species: Usnea taylorii and Pseudocyphellaria crocata. A controlled feeding experiment was designed to understand how the snail copes with the unpalatable and/or toxic compounds produced by these lichen species. The occurrence of two snail ecophenotypes, represented by a mineral shell and an organic shell, led to address the question of a metabolic response specific to the phenotype. Snails were fed for two months with one of these lichens and the chemical profiles of biological samples of N. hookeri (i.e., crop, digestive gland, intestine, and feces) were established by HPLC-DAD-MS and compared to that of the lichens. N. hookeri appears as a generalist lichen feeder able to consume toxic metabolite-containing lichens, independently of the ecophenotype. The digestive gland did not sequester lichen metabolites. The snail metabolism might be based on four non-exclusive processes according to the concerned metabolites (avoidance, passive transport, hydrolysis, and excretion).

The LC/MS analysis was carried out using Advion Expression® CMS ESI.

Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel

M.R. Chae, S.J. Kang, K.P. Lee, B.R. Choi, H.K. Kim, J.K. Park, C.Y. Kim, S.W. Lee

Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30 μg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9 ± 1.7 to 0.6 ± 0.1 sec (n = 6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1 ± 5.8 to 5.2 ± 8.7 mV (n = 6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10 μm Zn2+ , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility.

The LC/MS analysis was carried out using Advion Expression® CMS.

Inhibitory Activity of Halogenated 3-Benzylidenechroman-4-ones Against Lipopolysaccharide-stimulated Reactive Oxygen Species Production in RAW 264.7 Macrophages

Til Bahadur Thapa Magar, Tara Man Kadayat, Hye Jin Oh, Pil-Hoon Park, Eun-Seok Lee

Chromanone-containing compounds have been reported to possess several important biological activities. As a part of our continuing effort for discovering potent anti-inflammatory agents, a series of halogen-containing 3-benzylidenechroman-4-ones (1–15) were synthesized, and evaluated for their inhibitory effect on lipopolysaccharide (LPS)-stimulated reactive oxygen species (ROS) production in RAW 264.7 macrophages. Compounds 4 and 10 exhibited significant inhibitory activity (IC50  = 5.09 ± 1.27 and 5.11 ± 0.51 μM, respectively) against LPS-stimulated ROS production in RAW 264.7 macrophages.

The LC/MS analysis was carried out using Advion Expression® CMS ESI.

Iron Bioavailability Studies of the First Generation of Iron-Biofortified Beans Released in Rwanda

Raymond Glahn, Elad Tako, Jonathan Hart, Jere Haas, Mercy Lung’aho, Steve Beebe

This paper represents a series of in vitro iron (Fe) bioavailability experiments, Fe content analysis and polyphenolic profile of the first generation of Fe biofortified beans (Phaseolus vulgaris) selected for human trials in Rwanda and released to farmers of that region. The objective of the present study was to demonstrate how the Caco-2 cell bioassay for Fe bioavailability can be utilized to assess the nutritional quality of Fe in such varieties and how they may interact with diets and meal plans of experimental studies. Furthermore, experiments were also conducted to directly compare this in vitro approach with specific human absorption studies of these Fe biofortified beans. The results show that other foods consumed with beans, such as rice, can negatively affect Fe bioavailability whereas potato may enhance the Fe absorption when consumed with beans. The results also suggest that the extrinsic labelling approach to measuring human Fe absorption can be flawed and thus provide misleading information. Overall, the results provide evidence that the Caco-2 cell bioassay represents an effective approach to evaluate the nutritional quality of Fe-biofortified beans, both separate from and within a targeted diet or meal plan.

The LC/MS analysis was carried out using Advion Expression® CMS ESI.

Direct analysis of volatile organic compounds in foods by headspace extraction atmospheric pressure chemical ionisation mass spectrometry

P. Perez-Hurtado, E. Palmer, T. Owen, C. Aldcroft, M.H. Allen, J. Jones,,C.S. Creaser, M.R. Lindley, M.A. Turner, J.C. Reynolds

The rapid screening of volatile organic compounds (VOCs) by direct analysis has potential applications in the areas of food and flavour science. Currently, the technique of choice for VOC analysis is gas chromatography/mass spectrometry (GC/MS). However, the long chromatographic run times and elaborate sample preparation associated with this technique have led a movement towards direct analysis techniques, such as selected ion flow tube mass spectrometry (SIFT-MS), proton transfer reaction mass spectrometry (PTR-MS) and electronic noses. The work presented here describes the design and construction of a Venturi jet-pump-based modification for a compact mass spectrometer which enables the direct introduction of volatiles for qualitative and quantitative analysis.

The MS analysis was carried out using Advion Expression® CMS with vAPCI.

Explosive and pollutant TNP detection by structurally flexible SOFs: DFT-D3, TD-DFT study and in vitro recognition

Pritam Ghosh, Partha Roy, Anata Ghosh, Saibal Jana, Naresh Chandra Murmu, Subhra Kanti Mukhopadhyay, Priyabrata Banerjee

Explosive and Pollutant Nitro Aromatics (epNACs) like 2,4,6-trinitrophenol (TNP) has been detected from various surface water specimens by luminescent Schiff base Organic Frameworks (SOFs) by fluorometric method. Fluorescence intensity of the receptor SOFs have been quenched in presence of TNP due to RET and ICT, which has been confirmed through solid and solution level spectroscopic studies like FT-IR, 1H-NMR, fluorescence titration. Modern DFT (DFT-D3) calculations of the possible host guest conformers have been performed for exploration of plausible route of interaction between receptor and epNACs. The outcome of theoretical calculations is in line with experimental findings where TNP and receptor conformation mimic parallel displaced type π- π interaction. TD-DFT has been executed with both receptor and receptor ···TNP adduct, the fluorescence quenching is in line with experimental outcome. Limit of TNP detection has been found as low as 5 μM with 2.97×104 M-1 as binding constant. In real time stepping, TNP as mutagenic agent for aquatic life has been detected inside prokaryotic cells like candidia albicans in ppm level.

The MS analysis was carried out using Advion Expression® CMS ESI.

An evaluation of central penetration from a peripherally administered oxytocin receptor selective antagonist in nonhuman primates

Aaron L. Smith, Hasse Walum, Fawn Connor-Stroud, Sara M. Freeman, Kiyoshi Inoue, Lisa A. Parr, Mark M. Goodman, Larry J. Young

The physiology of the oxytocin receptor has increasingly become a focus of scientific investigation due to its connection with social behavior and psychiatric disorders with impairments in social funciton. Experimental utilization of small molecule and peptide antagonists for the oxytocin receptor has played a role in deciphering these biological and social behavior connections in rodents. Described herein is the evaluation of a potent and selective oxytocin receptor antagonist, ALS-I-41, and details to consider for its use in nonhuman primate behavioral pharmacology experiments utilizing intranasal or intramuscular administration. The central nervous system penetration and rate of metabolism of ALS-I-41 was investigated via mass spectroscopy analysis of cerebrospinal fluid and plasma in the rhesus macaque after intranasal and intramuscular administration. Positron emission tomography was also utilized with [18F] ALS-I-41 in a macaque to verify observed central nervous system (CNS) penetration and to further evaluate the effects of administration rate on CNS penetration of Sprague-Dawley rats in comparison to previous studies.

The LC/MS analysis was carried out using Advion Expression® CMS.

Hydrazine functionalized probes for chromogenic and fluorescent ratiometric sensing of pH and F−: experimental and DFT studies

Additi Roy Chowdhury, Amita Mondal, Biswajit Gopal Roy, Jagadeesh C. Bose K, Sudit Mukhopadhyaye and Priyabrata Banerjee

Two novel hydrazine based sensors, BPPIH (N1, N3-bis(perfluorophenyl)isophthalohydrazide) and BPBIH (N1′, N3′-bis(perfluorobenzylidene)isophthalohydrazide), are presented here. BPPIH is found to be a highly sensitive pH sensor in the pH range 5.0 to 10.0 in a DMSO–water solvent mixture with a pKa value of 9.22. Interesting optical responses have been observed for BPPIH in the above mentioned pH range. BPBIH on the other hand turns out to be a less effective pH sensor in the above mentioned pH range. The increase in fluorescence intensity at a lower pH for BPPIH was explained by using density functional theory. The ability of BPPIH to monitor the pH changes inside cancer cells is a useful application of the sensor as a functional material. In addition fluoride (F−) selectivity studies of these two chemosensors have been performed and show that between them, BPBIH shows greater selectivity towards F−. The interaction energy calculated from the DFT-D3 supports the experimental findings. The pH sensor (BPPIH) can be further interfaced with suitable circuitry interfaced with desired programming for ease of access and enhancement of practical applications.

The MS analysis was carried out using Advion Expression® CMS ESI.

Fast Identification of Artificial Vanilla Flavor in Vanilla Extract by ASAP® Compact Mass Spectrometry

Vanilla extract is a baking staple for cookies, cupcakes and more – but not all vanilla extract is created equal. Pure vanilla extract is made from the vanilla bean pod of the Vanilla planifolia plant. Imitation vanilla is derived from ethyl vanillin, which can be synthesized without the use of any vanilla beans at all. While pure vanilla extract may seem like the obvious choice, it does come at a price, making the imitation product a viable option in supermarkets across the world. But it begs the question: what is the chemical difference, and are products marketed as “pure” truly authentic?

In this screening assay, the expression® CMS coupled with the Atmospheric Solids Analysis Probe (ASAP®) is used to quickly identify the presence of artificial vanilla, as well as confirm the purity of products marketed as such. This technique provides a fast and easy method for purity analysis.

University of Birmingham, Mass Spectrometry Facility

Q: WHAT IS THE FOCUS OF YOUR LAB’S RESEARCH?

A: We provide our expertise, combining chromatography and mass spectrometry, as well as other Analytical techniques within the Facility. This facilitates and assists in the characterization of compounds synthesized in the Chemistry Department and a number of other departments within the University of Birmingham in addition to work for industrial collaborators.

Q: WHAT WAS YOUR PREVIOUS WORK FLOW OR CHALLENGES?

A: We needed a system capable of analysing reaction mixtures to assist in the identification of compounds of interest with minimal sample isolation required and provide a relatively quick identification of the compounds. The Advion TLC-MS has fully met these requirements, in terms of the ease of use as a walk-up instrument. This has greatly assisted in providing separation conditions in purification method development, as well as, a useful tool in simple reaction monitoring. The system has enabled us to directly obtain good quality data from complex mixtures of compounds. This has been of great benefit to research students across Chemistry, Chemical Engineering, Biochemistry and Pharmacology Departments who have all used the Advion TLC-MS. The major advantage of the Advion TLC-MS is the ability for the users to visualise the spot on the TLC that is being analysed by the mass spectrometer. If the sample requires further clean-up, the TLC conditions provide the initial starting point for upscaling purification using preparative columns.

Q: WHY DID YOU INCORPORATE THE EXPRESSION CMS INTO YOUR LABORATORY?

A: The Advion TLC-MS system is setup using an HPLC pump plumbed directly into the TLC sampler which is then plumbed back through the HPLC autosampler and finally into the Advion expression CMS. This setup has benefitted users to simply run a TLC and perform the MS analysis which takes just a few minutes and they have a lot of useful information about their reaction, product identity, impurities etc. The HPLC autosampler act as a method of directly delivering fractions collected from preparative HPLC allowing the users to quickly identify the fraction of interest. The feedback we have had is that by using the Advion TLC-MS the users are now spending significantly more time running reactions, increasing research output by spending less time isolating and characterizing each component in their reaction mixtures.