TmaDB: a repository for tissue microarray data

Background: Tissue microarray (TMA) technology has been developed to facilitate large, genome-scale molecular pathology studies. This technique provides a high-throughput method for analyzing a large cohort of clinical specimens in a single experiment thereby permitting the parallel analysis of molecular alterations ( at the DNA, RNA, or protein level) in thousands of tissue specimens. As a vast quantity of data can be generated in a single TMA experiment a systematic approach is required for the storage and analysis of such data. Description: To analyse TMA output a relational database ( known as TmaDB) has been developed to collate all aspects of information relating to TMAs. These data include the TMA construction protocol, experimental protocol and results from the various immunocytological and histochemical staining experiments including the scanned images for each of the TMA cores. Furthermore the database contains pathological information associated with each of the specimens on the TMA slide, the location of the various TMAs and the individual specimen blocks ( from which cores were taken) in the laboratory and their current status i.e. if they can be sectioned into further slides or if they are exhausted. TmaDB has been designed to incorporate and extend many of the published common data elements and the XML format for TMA experiments and is therefore compatible with the TMA data exchange specifications developed by the Association for Pathology Informatics community. Finally the design of the database is made flexible such that TMA experiments from several types of cancer can be stored in a single database, which incorporates the national minimum data set required for pathology reports supported by the Royal College of Pathologists (UK). Conclusion: TmaDB will provide a comprehensive repository for TMA data such that a large number of results from the numerous immunostaining experiments can be efficiently compared for each of the TMA cores. This will allow a systematic, large-scale comparison of tumour samples to facilitate the identification of gene products of clinical importance such as therapeutic or prognostic markers. In addition this work will contribute to the establishment of a standard for reporting TMA data analogous to MIAME in the description of microarray dat

Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide (TMAO) by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate inter-subunit electron transfer between the Rieske centre and the catalytic mononuclear iron centre. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Together, our study reveals the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assigns the role of this novel group of Rieske-type proteins in microbial carnitine metabolism

Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for ‘metabolic retroversion’

Dietary sources of methylamines such as choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It is recognized that the gut microbiota is able to convert choline to TMA in a fermentation-like process. Similarly, PC and carnitine are converted to TMA by the gut microbiota. It has been suggested that TMAO is subject to ‘metabolic retroversion’ in the gut (i.e. it is reduced to TMA by the gut microbiota, with this TMA being oxidized to produce TMAO in the liver). Sixty-six strains of human faecal and caecal bacteria were screened on solid and liquid media for their ability to utilize trimethylamine N-oxide (TMAO), with metabolites in spent media profiled by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. Enterobacteriaceae produced mostly TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria (enterococci, streptococci, bifidobacteria) produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA from TMAO. The presence of TMAO in media increased the growth rate of Enterobacteriaceae; while it did not affect the growth rate of lactic acid bacteria, TMAO increased the biomass of these bacteria. The positive influence of TMAO on Enterobacteriaceae was confirmed in anaerobic, stirred, pH-controlled batch culture fermentation systems inoculated with human faeces, where this was the only bacterial population whose growth was significantly stimulated by the presence of TMAO in the medium. We hypothesize that dietary TMAO is used as an alternative electron acceptor by the gut microbiota in the small intestine/proximal colon, and contributes to microbial population dynamics upon its utilization and retroversion to TMA, prior to absorption and secondary conversion to TMAO by hepatic flavin-containing monooxygenases. Our findings support the idea that oral TMAO supplementation is a physiologically-stable microbiota-mediated strategy to deliver TMA at the gut barrier

Identification of the dimethylamine-trimethylamine complex in the gas phase

We have identified the dimethylamine-trimethylamine complex (DMA-TMA) at room temperature in the gas phase. The Fourier transform infrared (FTIR) spectrum of DMA-TMA in the NH-stretching fundamental region was obtained by spectral subtraction of spectra of each monomer. Explicitly correlated coupled cluster calculations were used to determine the minimum energy structure and interaction energy of DMA-TMA. Frequencies and intensities of NH-stretching transitions were also calculated at this level of theory with an anharmonic oscillator local mode model. The fundamental NH-stretching intensity in DMA-TMA is calculated to be approximately 700 times larger than that of the DMA monomer. The measured and calculated intensity is used to determine a room temperature equilibrium constant of DMA-TMA of 1.7 × 10⁻³ atm⁻¹ at 298 K

Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of \u3cem\u3eChlamydia trachomatis\u3c/em\u3e Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription-Mediated Amplification: Identification of Urinary Substances Associated with Inhibition and Removal of Inhibitory Activity

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P = 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odd ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and -70°C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors

Evaluation of flight efficiency for Stockholm Arlanda Airport using OpenSky Network data

Identification of causes of the delays within transition airspace is an important step in evaluating performance of the Terminal Maneuvering Area (TMA) Air Navigation Services: without knowing the current performance levels, it is difficult to identify which areas could be improved. Inefficient vertical profiles within TMA and deviations from the optimal flight paths due to bad weather conditions are the main sources of performance decline. In this work, we analyse punctuality and vertical efficiency of Stockholm Arlanda airport arrivals, and seek to quantify the fuel consumption impact associated with the inefficient vertical flight profiles within the Terminal Maneuvering Area (TMA). We use Opensky Network data for evaluation of the Stockholm Arlanda airport performance, comparing it to the DDR2 data provided by Eurocontol, outlining the advantages and disadvantages of both.Peer ReviewedPostprint (author's final draft

Proton Availability at the Air/Water Interface

The acidity of the water surface sensed by a colliding gas is determined in experiments in which the protonation of gaseous trimethylamine (TMA) on aqueous microjets is monitored by online electrospray mass spectrometry as a function of the pH of the bulk liquid (pH_(BLK)). TMAH^+ signal intensities describe a titration curve whose equivalence point at pH_(BLK) 3.8 is dramatically smaller than the acidity constant of trimethylammonium in bulk solution, pK_A(TMAH^+) = 9.8. Notably, the degree of TMA protonation above pH_(BLK) 4 is enhanced hundred-fold by submillimolar LiCl or NaCl and weakly inhibited at larger concentrations. Protonation enhancements are associated with the onset of significant direct kinetic solvent hydrogen isotope effects. Since TMA(g) can be protonated by H_2O itself only upon extensive solvent participation, we infer that H3O^+ emerges at the surface of neat water below pH_(BLK) 4

On site remediation of micropollutants from stormwater for reuse

University of Technology, Sydney. Faculty of Engineering and Information Technology.Water scarcity due to persistent drought is forcing the countries around the world to explore alternative fresh water resources. Australia is the driest inhabited continent, and has one of the most variable rainfall intensities in the world. In the last one hundred years Australia has suffered six major droughts and fifteen less severe droughts. The drought that commenced in 2001 has encouraged the harvesting of stormwater and re-use of water in order to lower the demand placed on municipal water supplies. Urban and industrial stormwater runoff has high potential as a reusable water resource, although it requires treatment due to the presence of several types of contaminants including inorganic ones that have adverse ecological impacts on receiving waters. The main aim of this research was to focus on the identification of contaminants of concern in Australian stormwater and provide suitable remediation solutions with effective onsite treatment practices such as biofiltration and adsorption/ ion exchange column techniques. Long term biofilter experiments were conducted with raw stormwater collected from a canal at Carlton, in Sydney. Anthracite and granular activated carbon (GAC) were used as a single filter media in biofilter columns. Media heights of 75 and 40 cm were used. The filter columns were operated at filtration velocities of 0.12 and 0.25 m/h. The removal efficiency for turbidity and DOC for the GAC filter media were found to be 75% and almost 100% respectively. The removal efficiency for the anthracite filter was much lower. Molecular weight distribution analysis showed an almost similar trend to the DOC removal. When compared to the anthracite filter media, the GAC biofilter removed a much larger range of organic compounds present in the stormwater. The GAC biofilter removed organic matter earlier as compared to the anthracite filter. Based on a limited sample of stormwater, the removal efficiency for phosphorus was upto 74% and that of nitrogen was up to 30%. In general, the GAC filter showed higher heavy metal removal efficiency than the anthracite filter. The removal of zinc, iron, lead and nickel were good, however, the concentration of heavy metals in the raw surface water sample was low. In another study, Organic matter removal from a diluted synthetic landfill leachate was studied using a GAC biofilter. This filter with a depth of 35cm was found to remove a significant amount of organic matter from the diluted synthetic landfill leachate. The experiments were conducted at low (0.2 m/h) and high (2 m/h) flow velocity through the GAC filter to represent insitu and exsitu biofiltration. The results showed that organic matter can be removed in a consistent manner for a long period of time. GAC bio-filtration led to a consistent TOC removal even after a long period of operation without the need to regenerate the activated carbon. Even after 30-50 days of continuous running, the organic removal efficiency from the GAC bio-filter was approximately 40% and 60% when high (2 m/hr) and low (0.2 m/hr) filtration velocities were used. It should be noted that the performance can be enhanced by using a larger filter depth which is the case in real situations. A study was conducted using three filters in series to evaluate the efficiency of removal of TOC, turbidity and nutrients from stormwater. The first filter system containing sand removed 70% turbidity and 6% TOC. The second filter system containing GAC removed 99% of the remaining TOC and 43% of the remaining turbidity. The use of a Purolite A520E filter media as the third stage filter was found to be capable of removing up to 89% of nitrate within 6 hours, however no phosphate removal was achieve. Therefore it is vital to have GAC and Purolite as a media in filtration system along with sand to achieve organic and nutrient removal. Combined removal of a mixture of heavy metals (Cd, Cu, Ni and Zn) and an oxy anion of Se (predominantly selenate species, SeO4-) from a synthetic stormwater sample was investigated by their sorption on hydrous ferric oxide (HFO) (5%), HFO+Ca(OH)2 (6%), and HFO+Ca(OH)2+MnO2 (7%) in columns containing 93-95% anthracite, conducted under seven intermittent runoffs (wetting and drying) each of 8 h duration within a 40 h period. The contaminants removal behaviour varied between the ions and between the sorbents as well as with flow rate and time. The removal efficiency was greater at a low flow rate (0.75 m/h) than at a high flow rate (1.5 m/h). At the initial period when Ca(OH)2 produced elevated pHs, the HFO only column removed less heavy metals but more Se than the HFO+Ca(OH)2 columns. With increased time when the pH effect of Ca(OH)2 became insignificant, the MnO2 in the column increased the removal of all contaminants. The removal efficiencies (%) at the low flow rate for Cd, Cu, Ni, Zn by the HFO+Ca(OH)2+MnO2 column, and Se by the HFO column were 95-100, 94-98, 88-99, 96-100, 92-94 for the 1st and 3rd runs and 61-76, 85-88, 51-69, 57-79, 82-88 for the 5th and 7th runs, respectively. Urban road-deposited sediments (RDS) are potential heavy metal pollution sources of both terrestrial and aquatic environments. A study was conducted to determine the heavy metals enrichments, their possible sources and potential bioavailability and mobility in RDS from nine sites along major motorways of Sydney, the largest city with highest road traffic density in Australia. The results showed that the mean total concentrations of metals in the RDS decreased in the order, Fe > Mn > Zn > Cu > Cr >Pb> Ni> Cd. The corresponding order in the background soils (minimally contaminated from roads) was Fe >Mn > Zn ~ Ni > Cu ~ Pb > Cr > Cd. Both the pollution index (PI) and metal enrichment factor (EF), which are comparative measures between contaminated and uncontaminated sites were highest for Cu and Zn suggesting that Cu and Zn inputs to RDS were mainly the likely result of brake and tyre wear, respectively. Cluster and correlation analyses showed that while the concentrations of these two metals were related in the soil, they were not correlated in the RDS. Low PI and EF values as well as the close inter-relationships of Fe, Mn, Cr and Ni in both RDS and soils, would suggest that these metals were derived mainly from natural sources. Metal fractionation data showed that 50-95% of Cr and Fe in RDS were present in the immobile and bio-unavailable residual fraction, whereas 15-65% of Zn was contained in the exchangeable fraction which is considered to be mobile and bioavailable. Significant quantities of Mn, Ni, Cu, Pb and Cd were detected in all fractions, although the residual fraction contained the majority in all instances. Copper was the only metal appreciably present (5-25%) in the organic fraction