Distribution of mercury in a tropical estuary (India) situated near a chloro-alkali plant

The distribution of mercury in water, sediment and some biological samples of the Rushikulya estuary, east coast of India were assessed during Jan-Dec. 1989. Both the dissolved plus acid leachable mercury contents in water and the sediment mercury discerned conspicuous spatial and seasonal fluctuations. Adsorption on to the suspended particulates was found to be the most likely mechanism for removal of mercury from the water column. Exchange of mercury from sediments to water was observed at high salinities (20-30x10-3). The residual mercury contents in the biological samples revealed that bio-accumulation by bottom-dwelling organisms are higher than the pelagic components.

Enzymatic reaction of the immobilized enzyme on porous silicon studied by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a matrix-free technique that allows for the direct desorption/ionization of low-molecular-weight compounds with little or no fragmentation of analytes. This technique has a relatively high tolerance for contaminants commonly found in biological samples. DIOS-MS has been applied to determine the activity of immobilized enzymes on the porous silicon surface. Enzyme activities were also monitored with the addition of a competitive inhibitor in the substrate solution. It is demonstrated that this method can be applied to the screening of enzyme inhibitors. Furthermore, a method for peptide mapping analysis by in situ digestion of proteins on the porous silicon surface modified by trypsin, combined with matrix-assisted laser desorption/ionization-time of flight-MS has been developed.

Investigation of Calmodulin-Peptide Interactions Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

In this report, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to study the binding interactions between calmodulin and two target peptides (melittin and substance P). Various matrix conditions were tested and the less acidic matrix DHAP and THAP were found to favor the survival of the intact calcium-calmodulin as well as the calmodulin-peptide complexes. However, the application of direct MALDI-MS to detect the intact complexes turned out to be very difficult due to the dissociation of the complexes and the formation of nonspecific aggregates. In contrast, the specific binding of the target peptides to calmodulin could be easily deduced using intensity-fading (IF) MALDI-MS. Compared with the nonbinding control, clear reduction in the ion abundances of the target peptides was observed with the addition of calmodulin.

Improvements in the selectivity of electrochemical detectors for liquid chromatography and flow injection analysis using the self-assembled n-alkanethiol monolayer-modified au electrode

4-Aminophenol (4-AP), paracetamol (PRCT), norepinephrine (NE), and dopamine (DA) (all somewhat hydrophobic compounds) were HPLC electrochemically detected while the signals from uric acid (UA) and ascorbic acid (AA) (both hydrophilic compounds at the pH studied) were minimized, taking advantage of the permselectivity of the self-assembled n-alkanethiol monolayer (C-10-SAM)-modified Au electrodes based on solute polarity, The effects of various factors, such as the chain length of the n-alkanethiol modifier, modifying time, and pH value, on the permeability of C-10-SAM coatings were examined, The calibration curves, linear response ranges, detection limits, and reproducibilities of the EC detector for 4-AP, PRCT, NE, and DA were obtained, The result shows that the EC detector can be applied in the chromatographic detection of 4-AP, PRCT, NE, and DA in urine, effectively removing the influence of UA and AA in high concentrations existing in biological samples. As a result, a great improvement in the selectivity of EC detectors has been achieved by using Au electrodes coated with neutral n-alkanethiol monolayer.

STM OF FOLDED AND UNFOLDED HEMOGLOBIN MOLECULES ELECTROCHEMICALLY DEPOSITED ON HIGHLY ORIENTED PYROLYTIC-GRAPHITE

The structural characterization of folded and unfolded haemoglobin has been performed by scanning tunnelling microscopy (STM) for the first time. STM images show an oval-shaped pattern for the folded structure of this protein, and moreover two dimers consisting of one haemoglobin molecule can be clearly discerned. The dimensions of a folded molecule were determined as 6.4 x 5.4 x 0.7 nm(3), which are in good agreement with the known size obtained from X-ray analysis. We have found that unfolding of haemoglobin molecules on the surface of highly oriented pyrolytic graphite (HOPG) can be achieved by electrochemical deposition. The STM analysis indicates clearly that the tertiary structure of the protein was lost by electrochemical deposition, and most of the haemoglobin molecules were almost fully extended and exhibited a twisted rope-like or a rod-like aggregated structure. Our investigation demonstrates the capability of the electrochemical method in denaturing this redox protein and in preparing stable biological samples for use in STM imaging.

Enriched accumulation and biotransformation of selenium in the edible seaweed Laminaria japonica

Accumulations of selenium in kelp Laminaria japonica cultured in seawater was achieved by adding selenite (Na2SeO3) with or without N-P (NaNO3 + NaH2PO4) nutrients at different concentrations. Biotransformation of selenium in the kelp was investigated through measuring the selenium of biological samples and different biochemical fractionations. The results showed that the optimal selenite-enrichment concentration is 200 mg L-1, which can allow the kelp to accumulate a total selenium content from 0.51 +/- 0.15 to 26.23 +/- 3.12 mug g(-1) of fresh weight (fw). Selenium composition analysis of kelp (control group) showed that selenium is present as organic selenium, which is up to 86.22% of the total selenium, whereas inorganic selenium is barely 4.85%. When L. japonica was exposed for 56 h in seawater containing 200 mg L-1 Na2SeO3, the organic selenium was 16.70 mug g(-1) of fw (68.23%) and inorganic selenium was 4.71 mug g(-1) of fw (19.26%). The capability of accumulation of selenium was further enhanced by adding N-P nutrients to the selenite-enriched medium. Total selenium is increased to be 33.65 mug g(-1) of fw at optimal concentration of N-P nutrient (150 mg L-1 NaNO3 and 25 mg L-1 NaH2PO4), whereas the inorganic selenium was not increased and remained at 4.597 mug g(-1) of fw (13.36%), and the increased part of selenium was organic selenium. This implied that kelp L. japonica could effectively transform inorganic selenium into organic selenium through metabolism.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Separation and detection of compounds in Honeysuckle by integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

A hyphenated method for the isolation and identification of components in a traditional Chinese medicine of Honeysuckle was developed. Ion-exchange chromatography (IEC) was chosen for the fractionation of Honeysuckle extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further analyzed by reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer (RPLC-APCI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes, respectively. It can be noted totally more than 117 components were detected by UV detector, APCI/MS and MALDI-TOF/MS in Honeysuckle extract except the, 145 components identified by MALDI-TOF/MS alone with this integrated approach, and 7 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS, respectively. The obtained analytical results not only indicated the approach of integration IEC fractionation with RPLC-APCI/MS and MALDI-TOF/MS is capable of analyzing complex samples, but also exhibited the potential power of the mass spectrometer in detection of low-mass compounds, such as traditional Chinese medicines (TCMs) and complex biological samples. (c) 2005 Elsevier B.V. All rights reserved.

Adaptive denoising in spectral analysis by genetic programming

Rowland, J.J. and Taylor, J. (2002). Adaptive denoising in spectral analysis by genetic programming. Proc. IEEE Congress on Evolutionary Computation (part of WCCI), May 2002. pp 133-138. ISBN 0-7803-7281-6

Silica bonded stationary phases: Innovative functionalization methods and novel chromatographic separations based on molecular recognition

The research work included in this thesis examines the synthesis, characterization and chromatographic evaluation of novel bonded silica stationary phases. Innovative methods of preparation of silica hydride intermediates and octadecylsilica using a “green chemistry” approach eliminate the use of toxic organic solvents and exploit the solvating power and enhanced diffusivity of supercritical carbon dioxide to produce phases with a surface coverage of bonded ligands which is comparable to, or exceeds, that achieved using traditional organic solvent-based methods. A new stationary phase is also discussed which displays chromatographic selectivity based on molecular recognition. Chapter 1 introduces the chemistry of silica stationary phases, the retention mechanisms and theories on which reversed-phase liquid chromatography and hydrophilic interaction chromatograpy are based, the art and science of achieving a well packed liquid chromatography column, the properties of supercritical carbon dioxide and molecular recognition chemistry. Chapter 2 compares the properties of silica hydride materials prepared using supercritical carbon dioxide as the reaction medium with those synthesized in an organic solvent. A higher coverage of hydride groups on the silica surface is seen when a monofunctional silane is reacted in supercritical carbon dioxide while trifunctional silanes result in a phase which exhibits different properties depending on the reaction medium used. The differing chromatographic behaviour of these silica hydride materials prepared using supercritical carbon dioxide and using organic solvent are explored in chapter 3. Chapter 4 focusses on the preparation of octadecylsilica using mono-, di- and trifunctional alkoxysilanes in supercritical carbon dioxide and in anhydrous toluene. The surface coverage of octadecyl groups, as calculated using thermogravimetric analysis and elemental analysis, is highest when a trifunctional alkoxysilane is reacted with silica in supercritical carbon dioxide. A novel silica stationary phase is discussed in chapter 5 which displays selectivity for analytes based on their hydrogen bonding capabilities. The phase is also highly selective for barbituric acid and may have a future application in the solid phase extraction of barbiturates from biological samples.

SplicerAV: a tool for mining microarray expression data for changes in RNA processing.

BACKGROUND: Over the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets. RESULTS: Data from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets. CONCLUSIONS: Using SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.

Irisin - a myth rather than an exercise-inducible myokine.

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Long-term effects of ocean warming on the prokaryotic community: evidence from the vibrios

The long-term effects of ocean warming on prokaryotic communities are unknown because of lack of historical data. We overcame this gap by applying a retrospective molecular analysis to the bacterial community on formalin-fixed samples from the historical Continuous Plankton Recorder archive, which is one of the longest and most geographically extensive collections of marine biological samples in the world. We showed that during the last half century, ubiquitous marine bacteria of the Vibrio genus, including Vibrio cholerae, increased in dominance within the plankton-associated bacterial community of the North Sea, where an unprecedented increase in bathing infections related to these bacteria was recently reported. Among environmental variables, increased sea surface temperature explained 45% of the variance in Vibrio data, supporting the view that ocean warming is favouring the spread of vibrios and may be the cause of the globally increasing trend in their associated diseases.

Operation of a free-electron laser from the extreme ultraviolet to the water window

We report results on the performance of a free-electron laser operating at a wavelength of 13.7 nm where unprecedented peak and average powers for a coherent extreme-ultraviolet radiation source have been measured. In the saturation regime, the peak energy approached 170 J for individual pulses, and the average energy per pulse reached 70 J. The pulse duration was in the region of 10 fs, and peak powers of 10 GW were achieved. At a pulse repetition frequency of 700 pulses per second, the average extreme-ultraviolet power reached 20 mW. The output beam also contained a significant contribution from odd harmonics of approximately 0.6% and 0.03% for the 3rd (4.6 nm) and the 5th (2.75 nm) harmonics, respectively. At 2.75 nm the 5th harmonic of the radiation reaches deep into the water window, a wavelength range that is crucially important for the investigation of biological samples.

Optimizing The Nasal Allergen Challenge Model For The Study Of Allergic Rhinitis

Background The Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) uses a Nasal Allergen Challenge (NAC) model to study the pathophysiology of AR and provides proof of concept for novel therapeutics. The NAC model needs to ensure optimal participant qualification, allergen challenge, clinical symptoms capture and biological samples collection. Repeatability of the protocol is key to ensuring unbiased efficacy analysis of novel therapeutics. The effect of allergen challenge on IL-33 gene expression and its relation to IL1RL1 receptor and cytokine secretion was investigated. Methods Several iterations of the NAC protocol was tested, comparing variations of qualifying criteria based on the Total Nasal Symptom Score (TNSS) and Peak Nasal Inspiratory Flow (PNIF). The lowest allergen concentration was delivered and TNSS and PNIF recorded 15 minutes later. Participants qualified if the particular criteria for the protocol were met, otherwise the next higher allergen concentration (4-fold increase), was administered until the targets were reached. Participants returned for a NAC visit and received varying allergen challenge concentrations depending on the protocol, TNSS/PNIF were recorded at 15 minutes, 30 minutes, 1 hour, and hourly up to 12 hours, a 24 hour time point was added in later iterations. Repeatability was evaluated using a 3-4week interval between screening, NAC1, and NAC2 visits. Various biomarker samples were collected. Results A combined TNSS and PNIF criterion was more successful in qualifying participants. The cumulative allergen challenge (CAC) protocol proved more reliable in producing a robust clinical and biomarker response. Repeatability of the CAC protocol was achieved with a 3-week interval between visits, on a clinical and biological basis. IL-33 cytokine is an important biomarker in initiating the inflammatory response in AR in humans. IL-33 and IL1RL1 expression might employ a negative feedback mechanism in human nasal epithelial cells. Comparing the clinical and biological response to ragweed vs cat allergen challenge, proved the CAC protocol’s suitability for use employing different allergens. Conclusion The AR-CIC’s CAC protocol is an effective method of studying AR, capable of generating measurable and repeatable clinical and biomarker responses, enabling better understanding of AR pathophysiology and ensuring that any change would be purely due to medication under investigation in a clinical trial setting.