High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography.

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important 'fingerprint' for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.

High-resolution accurate mass spectrometry as a technique for characterization of complex lysimeter leachate samples.

Lysimeter studies can be used to identify and quantify soil degradates of agrochemicals (metabolites) that have the potential to leach to groundwater. However, the apparent metabolic profile of such lysimeter leachate samples will often be significantly more complex than would be expected in true groundwater samples. This is particularly true for S-metolachlor, which has an extremely complex metabolic pathway. Consequently, it was not practically possible to apply a conventional analytical approach to identify all metabolites in an S-metolachlor lysimeter study, because there was insufficient mass to enable the use of techniques such as nuclear magnetic resonance. Recent advances in high-resolution accurate mass spectrometry, however, allow innovative screening approaches to characterize leachate samples to a greater extent than previously possible. Leachate from the S-metolachlor study was screened for accurate masses (±5 ppm of the nominal mass) corresponding to more than 400 hypothetical metabolite structures. A refined list of plausible metabolites was constructed from these data to provide a comprehensive description of the most likely metabolites present. The properties of these metabolites were then evaluated using a principal component analysis model, based on molecular descriptors, to visualize the entire chemical space and to cluster the metabolites into a number of subclasses. This characterization and principal component analysis evaluation enabled the selection of suitable representative metabolites that were subsequently used as exemplars to assess the toxicological relevance of the leachate as a whole. Environ Toxicol Chem 2016;35:1401-1412. © 2015 SETAC.

MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics.

Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data.

Metabolic differences in ripening of Solanum lycopersicum 'Ailsa Craig' and three monogenic mutants.

Application of mass spectrometry enables the detection of metabolic differences between groups of related organisms. Differences in the metabolic fingerprints of wild-type Solanum lycopersicum and three monogenic mutants, ripening inhibitor (rin), non-ripening (nor) and Colourless non-ripening (Cnr), of tomato are captured with regard to ripening behaviour. A high-resolution tandem mass spectrometry system coupled to liquid chromatography produced a time series of the ripening behaviour at discrete intervals with a focus on changes post-anthesis. Internal standards and quality controls were used to ensure system stability. The raw data of the samples and reference compounds including study protocols have been deposited in the open metabolomics database MetaboLights via the metadata annotation tool Isatab to enable efficient re-use of the datasets, such as in metabolomics cross-study comparisons or data fusion exercises.

Variation in chlorotoluron photodegradation rates as a result of seasonal changes in the composition of natural waters.

BACKGROUND: It is important to understand the degradation of organic molecules in surface waters to ensure that risk assessments, intended to prevent adverse effects on human health and the environment, are robust. One important degradation mechanism in surface waters is photodegradation. This process is generally studied in laboratory test systems, and the significance of the results is then extrapolated to the field. The aim of this work was to assess how fluctuations in the composition of surface water influence the photodegradation rate of chlorotoluron. RESULTS: Photodegradation DT(50) values in the lake (mean = 26.0 days) and pond (mean = 26.0 days) were significantly slower than in the river (mean = 6.8 days) and stream (mean = 7.3 days) samples. The DT(50) values in the pond and lake samples were similar to the direct photolysis value (mean = 28.6 days). Photodegradation was significantly faster in the stream and river samples, suggesting that indirect photolysis was significant in those waters. Principal component analysis indicated a strong inverse correlation between nitrate concentration and degradation rate. CONCLUSIONS: Nitrate concentration had a strong influence on the rate of photodegradation, with increasing nitrate concentrations sharply reducing the DT(50) . However, this effect was restricted to a narrow concentration range and levelled off quite quickly, such that further increases in the nitrate concentration had no significant effect on the rate of degradation. Extrapolating photodegradation rates of chlorotoluron from the laboratory to the field should be relatively straightforward, provided the nitrate concentrations in the waters are known.

A QC approach to the determination of day-to-day reproducibility and robustness of LC-MS methods for global metabolite profiling in metabonomics/metabolomics.

BACKGROUND: An approach to the determination of day-to-day analytical robustness of LC-MS-based methods for global metabolic profiling using a pooled QC sample is presented for the evaluation of metabonomic/metabolomic data. A set of 60 urine samples were repeatedly analyzed on five different days and the day-to-day reproducibility of the data obtained was determined. Multivariate statistical analysis was performed with the aim of evaluating variability and selected peaks were assessed and validated in terms of retention time stability, mass accuracy and intensity. RESULTS: The methodology enables the repeatability/reproducibility of extended analytical runs in large-scale studies to be determined, allowing the elimination of analytical (as opposed to biological) variability, in order to discover true patterns and correlations within the data. CONCLUSION: The day-to-day variability of the data revealed by this process suggested that, for this particular system, 3 days continuous operation was possible without the need for maintenance and cleaning. Variation was generally based on signal intensity changes over the 7-day period of the study, and was mainly a result of source contamination.

Wound drainage after metal-on-metal hip arthroplasty secondary to presumed delayed hypersensitivity reaction.

An emerging concern with metal-on-metal total hip arthroplasty is metal-induced hypersensitivity. Currently, this is a diagnosis of exclusion in patients with groin pain after metal-on-metal total hip arthroplasty. We describe a patient presenting nearly a year after arthroplasty with incisional drainage. Infection was presumed; but preoperative studies were nondefinitive, and the wound was explored. The operative cultures were negative; the histology revealed lymphocytic vasculitis. The patient recovered uneventfully after exchange to a metal polyethylene bearing couple. We believe that metal-induced hypersensitivity should be considered with draining wounds with this bearing couple if infection cannot be proven.

Does the mass spectrometer define the marker? A comparison of global metabolite profiling data generated simultaneously via UPLC-MS on two different mass spectrometers.

By coupling a single UPLC separation to two different types of mass spectrometer an unbiased comparison of the metabolite profiles produced by each instrument for a set of rat urine samples was obtained. The flow from the UPLC column was split equally and both streams of eluent were simultaneously directed to the inlets of the two mass spectrometers. Mass spectrometry on the eluent was undertaken using a triple quadrupole linear ion trap and a hybrid quadrupole time-of-flight mass spectrometer using both positive and negative ESI. Data from both mass spectrometers were subjected to multivariate statistical analysis, after applying the same data extraction software, and showed the same general pattern of correlation between the samples using both unsupervised and supervised methods of statistical analysis. Based on orthogonal partial least-squares discriminant analysis models a number of ions were recognized as "responsible" for the separation of the animal groups. From the peaks detected, and denoted as significant by the statistical analysis a number of ions were found to be unique to one data set or the other, a result which may have consequences for biomarker discovery and interlaboratory comparisons. The software package used for data analysis also had an effect on the outcome of the statistical analysis.

Target list building for volatile metabolite profiling of fruit.

Fruit flavour is the combination of numerous biochemicals: sugars for sweetness, acids for sourness and volatile metabolites for aroma. The objective of this study was to establish a method to develop a target list of statistically relevant compounds for the characterization of melon from non-targeted data, while preserving the profile information. Five different varieties were sampled (sampling 12 biological replicates from 12 plants) using dynamic headspace extraction, then analysed by gas chromatography-mass spectrometry in full scan mode. Using Metalign and SIMCA-P software the raw data was spectrally aligned and then subjected to principal component analysis (PCA). The principal component analysis plot showed good separation of the five varieties based on their full scan GC-MS profile. Mass spectral data points responsible for the differences between varieties were highlighted by further statistical analysis. The mass spectra were then reconstructed and the corresponding chemicals identified using library search or reference standards were available to create a new target component list. To validate the new target list, the initial data set was re-processed using the targeted approach and the results subjected again to principal component analysis. The two representations showed excellent agreement on the separation of the five varieties. The new target list obtained from this study can be applied to differentiate and characterize the volatile profile of melon varieties using a list of statistically significant compounds.

Bilateral prosthetic knee infection by Campylobacter fetus.

We present the first documented case of a bilateral prosthetic knee joint infection with Campylobacter fetus. Our patient's risk factors included age, the presence of prosthetic joints, and potential exposure through his contact with farm animals. It is important to be aware of the possibility of C fetus joint infections in high-risk patients who present with pain after total joint arthroplasty.

Early osteolysis associated with trunion-liner impingement.

Osteolysis is perhaps the biggest unsolved problem in joint arthroplasty. Many factors contribute to osteolysis, some of which cannot be controlled. It is imperative to minimize controllable factors contributing to osteolysis whenever possible. We report four cases of trunion impingement on an elevated rim liner, which may have contributed to early aggressive osteolysis. Although the use of an elevated liner may result in a lower incidence of postoperative instability, the trade-off in using such a device must be understood.

Success rate of modular component exchange for the treatment of an unstable total hip arthroplasty.

Hip instability is the leading cause of morbidity after total hip arthroplasty. Surgical strategies that have been used to eliminate recurrent instability include component revision, trochanteric advancement, or the use of constrained components. Between 1986 and 1997, 731 revision total hip arthroplasties were performed at our institution. A total of 29 patients underwent modular component exchange to treat hip instability. After revision surgery, 16 of 29 (55%) patients experienced redislocation. Nine (31% overall) patients dislocated repeatedly after modular component exchange. Five of the 9 patients who dislocated repeatedly (17% overall) ultimately required rerevision to obtain stability. Modular component exchange is an unpredictable procedure in definitively solving hip stability problems. The limitations of this procedure in treating this complex multifactorial problem must be understood by patient and surgeon alike.