Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Genome analysis and characterisation of the exopolysaccharide produced by Bifidobacterium longum subsp. longum 35624™

The Bifibobacterium longum subsp. longum 35624™ strain (formerly named Bifidobacterium longum subsp. infantis) is a well described probiotic with clinical efficacy in Irritable Bowel Syndrome clinical trials and induces immunoregulatory effects in mice and in humans. This paper presents (a) the genome sequence of the organism allowing the assignment to its correct subspeciation longum; (b) a comparative genome assessment with other B. longum strains and (c) the molecular structure of the 35624 exopolysaccharide (EPS624). Comparative genome analysis of the 35624 strain with other B. longum strains determined that the sub-speciation of the strain is longum and revealed the presence of a 35624-specific gene cluster, predicted to encode the biosynthetic machinery for EPS624. Following isolation and acid treatment of the EPS, its chemical structure was determined using gas and liquid chromatography for sugar constituent and linkage analysis, electrospray and matrix assisted laser desorption ionization mass spectrometry for sequencing and NMR. The EPS consists of a branched hexasaccharide repeating unit containing two galactose and two glucose moieties, galacturonic acid and the unusual sugar 6-deoxy-L-talose. These data demonstrate that the B. longum 35624 strain has specific genetic features, one of which leads to the generation of a characteristic exopolysaccharide.

Desorption of stearic acid upon surfactant adsorbed montmorillonite : a thermogravimetric study

Thermal analysis and differential thermal analysis offers a means of studying the desorption of acids such as stearic acid from clay surfaces. Both adsorption and chemisorption can be distinguished through the differences in the temperature of the mass losses. Increased adsorption is achievable by adsorbing onto a surfactant adsorbed montmorillonite. Stearic acid sublimes at 179 °C but when adsorbed upon montmorillonite sublimes at 207 and 248 °C. These mass loss steps are ascribed to the desorption of the stearic acid on the external surfaces of the organoclays and from the de-chemisorption from the surfactant held in the interlayer of the montmorillonite.

Effect of thermal treatment on adsorption-desorption of ammonia and sulfur dioxide on palygorskite : change of surface acid-alkali properties

Thermally activated Palygorskite (Pg) has been found to be a good adsorbent material for ammonia (NH3) and sulfur dioxide (SO2). This research investigated the effect of thermal treatment on pore structure and surface acid-alkali properties of Pg through the adsorption-desorption of NH3 and SO2. The results showed that, up to 200 °C, the adsorption of NH3 on Pg was significantly higher than SO2. This was due to NH3 being adsorbed in the internal surface of Pg and forming hydrogen bonds (H-bonds) with coordinated water. The increase in thermal treatment temp. from 150 to 550 °C, showed a gradual decrease in the no. of surface acid sites, while the no. of surface alk. sites increased from 200 to 400 °C. The change of surface acidity-alk. sites is due to the collapse of internal channels of Pg and desorption of different types of hydroxyls assocd. with the Pg structure.

Vacancy mediated desorption of hydrogen from a sodium alanate surface : An ab initio spin-polarized study

Ab initio spin-polarized density functional theory calculations are performed to explore the effect of single Na vacancy on NaAlH4(001) surface on the initial dehydrogenation kinetics. The authors found that two Al–H bond lengths become elongated and weakened due to the presence of a Na vacancy on the NaAlH4(001) surface. Spontaneous recombination from the surface to form molecular hydrogen is observed in the spin-polarized ab initio molecular dynamics simulation. The authors’ results indicate that surface Na vacancies play a critical role in accelerating the dehydrogenation kinetics in sodium alanate. The understanding gained here will aid in the rational design and development of complex hydride materials for hydrogen storage

Ab initio studies of hydrogen desorption from low index magnesium hydride surface

The low index Magnesium hydride surfaces, MgH2(0 0 1) and MgH2(1 1 0), have been studied by ab intio Density Functional Theory (DFT) calculations. It was found that the MgH2(1 1 0) surface is more stable than MgH2(0 0 1) surface, which is in good agreement with the experimental observation. The H2 desorption barriers vary depending on the crystalline surfaces that are exposed and also the specific H atom sites involved – they are found to be generally high, due to the thermodynamic stability of the MgH2 system, and are larger for the MgH2(0 0 1) surface. The pathway for recombinative desorption of one in-plane and one bridging H atom from the MgH2(1 1 0) surface was found to be the lowest energy barrier amongst those computed (172 KJ/mol) and is in good agreement with the experimental estimates.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

Combination therapy with gefitinib and rofecoxib in patients with platinum-pretreated relapsed non-small-cell lung cancer

Purpose: In non-small-cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) play major roles in tumorigenesis. This phase I/II study evaluated combined therapy with the EGFR tyrosine kinase inhibitor (TKI) gefitinib and the COX-2 inhibitor rofecoxib in platinum-pretreated, relapsed, metastatic NSCLC (n = 45). Patients and Methods: Gefitinib 250 mg/d was combined with rofecoxib (dose escalated from 12.5 to 25 to 50 mg/d through three cohorts, each n = 6). Because the rofecoxib maximum-tolerated dose was not reached, the 50 mg/d cohort was expanded for efficacy evaluation (n = 33). Results: Among the 42 assessable patients, there was one complete response (CR) and two partial responses (PRs) and 12 patients with stable disease (SD); disease control rate was 35.7% (95% CI, 21.6% to 52.0%). Median time to tumor progression was 55 days (95% CI, 47 to 70 days), and median survival was 144 days (95% CI, 103 to 190 days). In a pilot study, matrix-assisted laser desorption/ionization (MALDI) proteomics analysis of baseline serum samples could distinguish patients with an objective response from those with SD or progressive disease (PD), and those with disease control (CR, PR, and SD) from those with PD. The regimen was generally well tolerated, with predictable toxicities including skin rash and diarrhea. Conclusion: Gefitinib combined with rofecoxib provided disease control equivalent to that expected with single-agent gefitinib and was generally well tolerated. Baseline serum proteomics may help identify those patients most likely to benefit from EGFR TKIs. © 2007 by American Society of Clinical Oncology.

Differentiation of isomeric N-glycan structures by normal-phase liquid chromatography-MALDI-TOF/TOF tandem mass spectrometry

The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.

Characterisation of the ionic products arising from electron photodetachment of simple dicarboxylate dianions

Much of what we currently understand about the structure and energetics of multiply charged anions in the gas phase is derived from the measurement of photoelectron spectra of simple dicarboxylate dianions. Here we have employed a modified linear ion-trap mass spectrometer to undertake complementary investigations of the ionic products resulting from laser-initiated electron photodetachment of two model dianions. Electron photodetachment (ePD) of the \[M-2H](2-) dianions formed from glutaric and adipic acid were found to result in a significant loss of ion signal overall, which is consistent with photoelectron studies that report the emission of slow secondary electrons (Xing et al., 2010 \[201). The ePD mass spectra reveal no signals corresponding to the intact \[M-2H](center dot-) radical anions, but rather \[M-2H-CO2](center dot-) ions are identified as the only abundant ionic products indicating that spontaneous decarboxylation follows ejection of the first electron. Interestingly however, investigations of the structure and energetics of the \[M-2H-CO2](center dot-) photoproducts by ion-molecule reaction and electronic structure calculation indicate that (i) these ions are stable with respect to secondary electron detachment and (ii) most of the ion population retains a distonic radical anion structure where the radical remains localised at the position of the departed carboxylate moiety. These observations lead to the conclusion that the mechanism for loss of ion signal involves unimolecular rearrangement reactions of the nascent \[M-2H](center dot-) carbonyloxyl radical anions that compete favourably with direct decarboxylation. Several possible rearrangement pathways that facilitate electron detachment from the radical anion are identified and are computed to be energetically accessible. Such pathways provide an explanation for prior observations of slow secondary electron features in the photoelectron spectra of the same dicaboxylate dianions. (C) 2013 Elsevier B.V. All rights reserved.

Rapid quantification of free cholesterol in tears using direct insertion/electron ionization-Mass spectrometry

Purpose. To establish a simple and rapid analytical method, based on direct insertion/electron ionization-mass spectrometry (DI/EI-MS), for measuring free cholesterol in tears from humans and rabbits. Methods. A stable-isotope dilution protocol employing DI/EI-MS in selected ion monitoring mode was developed and validated. It was used to quantify the free cholesterol content in human and rabbit tear extracts. Tears were collected from adult humans (n = 15) and rabbits (n = 10) and lipids extracted. Results. Screening, full-scan (m/z 40-600) DI/EI-MS analysis of crude tear extracts showed that diagnostic ions located in the mass range m/z 350 to 400 were those derived from free cholesterol, with no contribution from cholesterol esters. DI/EI-MS data acquired using selected ion monitoring (SIM) were analyzed for the abundance ratios of diagnostic ions with their stable isotope-labeled analogues arising from the D6-cholesterol internal standard. Standard curves of good linearity were produced and an on-probe limit of detection of 3 ng (at 3:1 signal to noise) and limit of quantification of 8 ng (at 10:1 signal to noise). The concentration of free cholesterol in human tears was 15 ± 6 μg/g, which was higher than in rabbit tears (10 ± 5 μg/g). Conclusions. A stable-isotope dilution DI/EI-SIM method for free cholesterol quantification without prior chromatographic separation was established. Using this method demonstrated that humans have higher free cholesterol levels in their tears than rabbits. This is in agreement with previous reports. This paper provides a rapid and reliable method to measure free cholesterol in small-volume clinical samples. © 2013 The Association for Research in Vision and Ophthalmology, Inc.

The fragmentation pathways of protonated Amiton in the gas phase : towards the structural characterisation of organophosphorus chemical warfare agents by electrospray ionisation tandem mass spectrometry

Amiton (O,O-diethyl-S-[2-(diethylamino)ethyl]phosphorothiolate), otherwise known as VG, is listed in schedule 2 of the Chemical Weapons Convention (CWC) and has a structure closely related to VX (O-ethyl-S-(2-diisopropylamino)ethylmethylphosphonothiolate). Fragmentation of protonated VG in the gas phase was performed using electrospray ionisation ion trap mass spectrometry (ESI-ITMS) and revealed several characteristic product ions. Quantum chemical calculations provide the most probable structures for these ions as well as the likely unimolecular mechanisms by which they are formed. The decomposition pathways predicted by computation are consistent with deuterium-labeling studies. The combination of experimental and theoretical data suggests that the fragmentation pathways of VG and analogous organophosphorus nerve agents, such as VX and Russian VX, are predictable and thus ESI tandem mass spectrometry is a powerful tool for the verification of unknown compounds listed in the CWC. Copyright (c) 2006 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.