Using metabolomics to understand stress responses in Lactic Acid Bacteria and their applications in the food industry.

BACKGROUND: Lactic Acid Bacteria (LAB) are commonly used as starter cultures, probiotics, to produce lactic acid and other useful compounds, and even as natural preservatives. For use in any food product however, LAB need to survive the various stresses they encounter in the environment and during processing. Understanding these mechanisms may enable direction of LAB biochemistry with potential beneficial impact for the food industry. AIM OF REVIEW: To give an overview of the use of LAB in the food industry and then generate a deeper biochemical understanding of LAB stress response mechanisms via metabolomics, and methods of screening for robust strains of LAB. KEY SCIENTIFIC CONCEPTS OF REVIEW: Uses of LAB in food products were assessed and factors which contribute to survival and tolerance in LAB investigated. Changes in the metabolic profiles of LAB exposed to stress were found to be associated with carbohydrates, amino acids and fatty acid levels and these changes were proposed to be a result of the bacteria trying to maintain cellular homeostasis in response to external conditions and minimise cellular damage from reactive oxygen species. This correlates with morphological analysis which shows that LAB can undergo cell elongation and shortening, as well as thinning and thickening of cell membranes, when exposed to stress. It is proposed that these innate strategies can be utilised to minimise negative effects caused by stress through selection of intrinsically robust strains, genetic modification and/or prior exposure to sublethal stress. This work demonstrates the utility of metabolomics to the food industry.

Exploring the potential application of alternative nuclei in NMR based metabolomics.

INTRODUCTION: Nuclear magnetic resonance (NMR) is widely used in metabolomics but it focusses on 1H over other NMR-active nuclei. OBJECTIVES: To evaluate the potential of alternative NMR-sensitive nuclei to generate useful metabolomic data. METHOD: Proton, carbon, phosphorus, and nitrogen-based NMR metabolomics was undertaken on extracts from mint and European honey bee tissue. RESULTS: Carbon NMR provided useful information but required larger sample sizes. Phosphorus produced overlapping peaks in one dimensional (1D) analysis but showed potential in 2D experiments. 15N NMR was found to not be sensitive enough for general metabolomic work. CONCLUSIONS: Alternative NMR active nuclei are useful for metabolomics.

Is substrate choice an overlooked variable in ecotoxicology experiments?

It is crucial to understand the effects caused by experimental parameters such as temperature, light, and food type on lab and field-based ecotoxicology experiments, as these variables, and combinations thereof, can affect results. The type of substrate used in exposure experiments, however, is generally assumed to have no effect. This may not always be correct. The metabolic changes in the freshwater crustacean, Austrochiltonia subtenuis exposed to copper, using three common substrates, gauze; toilet paper; and cellulose were investigated. Substrate alone did not affect survival, but each substrate elicited a different metabolic response and adult and juvenile amphipods had different substrate preferences. Several classes of metabolites were shown to change in response to different substrates and toxicant. These included disaccharides, monosaccharides, fatty acids, and tricarboxylic acid cycle intermediates. The results illustrate that metabolomic responses can differ in response to experimental factors that were previously thought not to be significant. In fact, our data indicate that substrate should be viewed as an experimental factor as important to control for as more well-known confounders such as temperature or food, thus challenging the current paradigm. Assuming substrate type has no effect on the experiment could potentially lead to errors in contaminant toxicity assessments. We propose that ideal good practise would be that all experimental factors should be evaluated for their potential influence on metabolomic profiles prior to contaminant response experiments being undertaken.

Vitamin C measurement in critical illness: challenges, methodologies and quality improvements.

Background There is renewed interest in high-dose vitamin C interventions in clinical medicine due to its antioxidant properties, safe use and cost-effectiveness. Yet, randomised control trials (RCTs) employing these interventions are failing to include robust analytical methodology and proper sample handling and processing techniques. Consequently, comparisons between studies becomes impossible as there is no metrological traceability and results may be prone to pre-analytical errors. Content Through published vitamin C stability studies, method comparison papers and data from vitamin C external quality assurance programs, an assessment was made on the functionality of current methods for critically ill patient samples. Summary Data was obtained from two external quality assurance programs, two papers assessing sample stability and interlaboratory agreement and a publication on vitamin C method comparisons. A shift from spectrophotometric and enzymatic methodologies to high performance liquid chromatography (HPLC) greatly improved the variability and interlaboratory agreement. Therefore, the current analytical performance of vitamin C HPLC methodologies are acceptable for the requirements of a high-dose vitamin C RCTs. Outlook Recommendations across the total testing process of vitamin C have been provided to improve the quality of the results. The harmonisation of sample handling and processing procedures will further improve the reliability of current analytical methodologies.

Using metabolomics to assess the sub-lethal effects of zinc and boscalid on an estuarine polychaete worm over time.

INTRODUCTION: Zinc is a heavy metal commonly detected in urban estuaries around Australia. Boscalid is a fungicide found in estuaries, both in water and sediment, it enters the system predominantly through agricultural run-off. Zinc is persistent while boscalid breaks down, with a half-life of 108 days. Both contaminants are widely distributed and their effects on ecosystems are not well understood. OBJECTIVES: The aim of this study was to determine the metabolite changes in Simplisetia aequisetis (an estuarine polychaete) following laboratory exposure to a sub-lethal concentration of zinc or boscalid over a 2-week period. METHODS: Individuals were collected at six time points over a 2-week period. Whole polychaete metabolites were extracted and quantified using a multi-platform approach. Polar metabolites were detected using a semi-targeted GC-MS analysis and amine containing compounds were analysed using a targeted LC-MS analysis. Total lipid energy content was also analysed for Simplisetia aequisetis. RESULTS: The pathways that responded to zinc and boscalid exposure were alanine, aspartate and glutamate metabolism (AAG); glycine, serine and threonine metabolism (GST) and metabolites associated with the tricarboxylic acid cycle (TCA). Results showed that changes in total abundance of some metabolites could be detected as early as 24-h exposure. Changes were detected in the metabolites before commonly used total lipid energy assays identified effects. CONCLUSION: A multi-platform approach provided a holistic overview of the metabolomic response to contaminants in polychaetes. This approach shows promise to be used in biomonitoring programs to provide early diagnostic indicators of contamination and exposure.

Illuminating the dark metabolome to advance the molecular characterisation of biological systems.

BACKGROUND: The latest version of the Human Metabolome Database (v4.0) lists 114,100 individual entries. Typically, however, metabolomics studies identify only around 100 compounds and many features identified in mass spectra are listed only as 'unknown compounds'. The lack of ability to detect all metabolites present, and fully identify all metabolites detected (the dark metabolome) means that, despite the great contribution of metabolomics to a range of areas in the last decade, a significant amount of useful information from publically funded studies is being lost or unused each year. This loss of data limits our potential gain in knowledge and understanding of important research areas such as cell biology, environmental pollution, plant science, food chemistry and health and biomedical research. Metabolomics therefore needs to develop new tools and methods for metabolite identification to advance as a field. AIM OF REVIEW: In this critical review, some potential issues with metabolite identification are identified and discussed. New and novel emerging technologies and tools which may contribute to expanding the number of compounds identified in metabolomics studies (thus illuminating the dark metabolome) are reviewed. The aim is to stimulate debate and research in the molecular characterisation of biological systems to drive forward metabolomic research. KEY SCIENTIFIC CONCEPTS OF REVIEW: The work specifically discusses dynamic nuclear polarisation nuclear magnetic resonance spectroscopy (DNP-NMR), non-proton NMR active nuclei, two-dimensional liquid chromatography (2DLC) and Raman spectroscopy (RS). It is suggested that developing new methods for metabolomics with these techniques could lead to advances in the field and better characterisation of biological systems.

Review of recent developments in GC-MS approaches to metabolomics-based research.

BACKGROUND: Metabolomics aims to identify the changes in endogenous metabolites of biological systems in response to intrinsic and extrinsic factors. This is accomplished through untargeted, semi-targeted and targeted based approaches. Untargeted and semi-targeted methods are typically applied in hypothesis-generating investigations (aimed at measuring as many metabolites as possible), while targeted approaches analyze a relatively smaller subset of biochemically important and relevant metabolites. Regardless of approach, it is well recognized amongst the metabolomics community that gas chromatography-mass spectrometry (GC-MS) is one of the most efficient, reproducible and well used analytical platforms for metabolomics research. This is due to the robust, reproducible and selective nature of the technique, as well as the large number of well-established libraries of both commercial and 'in house' metabolite databases available. AIM OF REVIEW: This review provides an overview of developments in GC-MS based metabolomics applications, with a focus on sample preparation and preservation techniques. A number of chemical derivatization (in-time, in-liner, offline and microwave assisted) techniques are also discussed. Electron impact ionization and a summary of alternate mass analyzers are highlighted, along with a number of recently reported new GC columns suited for metabolomics. Lastly, multidimensional GC-MS and its application in environmental and biomedical research is presented, along with the importance of bioinformatics. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is to both highlight and provide an update on GC-MS analytical techniques that are common in metabolomics studies. Specific emphasis is given to the key steps within the GC-MS workflow that those new to this field need to be aware of and the common pitfalls that should be looked out for when starting in this area.

Vitamin B1 in critically ill patients: needs and challenges.

BACKGROUND: Thiamine has a crucial role in energy production, and consequently thiamine deficiency (TD) has been associated with cardiac failure, neurological disorders, oxidative stress (lactic acidosis and sepsis) and refeeding syndrome (RFS). This review aims to explore analytical methodologies of thiamine compound quantification and highlight similarities, variances and limitations of current techniques and how they may be relevant to patients. CONTENT: An electronic search of Medline, PubMed and Embase databases for original articles published in peer-reviewed journals was conducted. MethodsNow was used to search for published analytical methods of thiamine compounds. Keywords for all databases included "thiamine and its phosphate esters", "thiamine methodology" and terms related to critical illness. Enquiries were also made to six external quality assurance (EQA) programme organisations for the inclusion of thiamine measurement. SUMMARY: A total of 777 published articles were identified; 122 were included in this review. The most common published method is HPLC with florescence detection. Two of the six EQA organisations include a thiamine measurement programme, both measuring only whole-blood thiamine pyrophosphate (TPP). No standard measurement procedure for thiamine compound quantification was identified. OUTLOOK: Overall, there is an absence of standardisation in measurement methodologies for thiamine in clinical care. Consequently, multiple variations in method practises are prohibiting the comparison of study results as they are not traceable to any higher order reference. Traceability of certified reference materials and reference measurement procedures is needed to provide an anchor to create the link between studies and help bring consensus on the clinical importance of thiamine.

A candidate reference method using ICP-MS for sweat chloride quantification.

BACKGROUND: The aim of the study was to develop a method for sweat chloride (Cl) quantification using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to present to the Joint Committee for Traceability in Laboratory Medicine (JCTLM) as a candidate reference method for the diagnosis of cystic fibrosis (CF). METHODS: Calibration standards were prepared from sodium chloride (NaCl) to cover the expected range of sweat Cl values. Germanium (Ge) and scandium (Sc) were selected as on-line (instrument based) internal standards (IS) and gallium (Ga) as the off-line (sample based) IS. The method was validated through linearity, accuracy and imprecision studies as well as enrolment into the Royal College of Pathologists of Australasia Quality Assurance Program (RCPAQAP) for sweat electrolyte testing. RESULTS: Two variations of the ICP-MS method were developed, an on-line and off-line IS, and compared. Linearity was determined up to 225 mmol/L with a limit of quantitation of 7.4 mmol/L. The off-line IS demonstrated increased accuracy through the RCPAQAP performance assessment (CV of 1.9%, bias of 1.5 mmol/L) in comparison to the on-line IS (CV of 8.0%, bias of 3.8 mmol/L). Paired t-tests confirmed no significant differences between sample means of the two IS methods (p=0.53) or from each method against the RCPAQAP target values (p=0.08 and p=0.29). CONCLUSIONS: Both on and off-line IS methods generated highly reproducible results and excellent linear comparison to the RCPAQAP target results. ICP-MS is a highly accurate method with a low limit of quantitation for sweat Cl analysis and should be recognised as a candidate reference method for the monitoring and diagnosis of CF. Laboratories that currently practice sweat Cl analysis using ICP-MS should include an off-line IS to help negate any pre-analytical errors.

Metal and metalloid containing natural products and a brief overview of their applications in biology, biotechnology and biomedicine.

Bioinorganic natural product chemistry is a relatively unexplored but rapidly developing field with enormous potential for applications in biology, biotechnology (especially in regards to nanomaterial development, synthesis and environmental cleanup) and biomedicine. In this review the occurrence of metals and metalloids in natural products and their synthetic derivatives are reviewed. A broad overview of the area is provided followed by a discussion on the more common metals and metalloids found in natural sources, and an overview of the requirements for future research. Special attention is given to metal hyperaccumulating plants and their use in chemical synthesis and bioremediation, as well as the potential uses of metals and metalloids as therapeutic agents. The potential future applications and development in the field are also discussed.

The effect of a high-roughage diet on the metabolism of aromatic compounds by rumen microbes: a metagenomic study using Mehsani buffalo (Bubalus bubalis).

In developing countries, livestock are often fed a high-lignin, low-nutrient diet that is rich in aromatic compounds. It is therefore important to understand the structure of the microbial community responsible for the metabolism of these substances. A metagenomic analysis was therefore carried out to assess the microbial communities associated with the liquid and solid fractions of rumen biomaterial from domestic Mehsani buffalo (Bubalus bubalis) fed with varying proportions of roughage. The experimental design consisted of three feeding regimes (50, 75 and 100 % roughage) and two roughage types (green and dry). Genes associated with aromatic compound degradation were assessed via high-throughput DNA sequencing. A total of 3914.94 Mb data were generated from all treatment groups. Genes coding for functional responses associated with aromatic compound metabolism were more prevalent in the liquid fraction of rumen samples than solid fractions. Statistically significant differences (p < 0.05) were also observed between treatment groups. These differences were dependent on the proportion of roughage fed to the animal, with the type of roughage having little effect. The genes present in the highest abundance in all treatment groups were those related to aromatic compound catabolism. At the phylum level, Bacteroidetes were dominant in all treatments closely followed by the Firmicutes. This study demonstrates the use of feed type to selectively enrich microbial communities capable of metabolizing aromatic compounds in the rumen of domestic buffalo. The results may help to improve nutrient utilization efficiency in livestock and are thus of interest to farming industries, particularly in developing countries, worldwide.

A Review of Analytical Techniques and Their Application in Disease Diagnosis in Breathomics and Salivaomics Research.

The application of metabolomics to biological samples has been a key focus in systems biology research, which is aimed at the development of rapid diagnostic methods and the creation of personalized medicine. More recently, there has been a strong focus towards this approach applied to non-invasively acquired samples, such as saliva and exhaled breath. The analysis of these biological samples, in conjunction with other sample types and traditional diagnostic tests, has resulted in faster and more reliable characterization of a range of health disorders and diseases. As the sampling process involved in collecting exhaled breath and saliva is non-intrusive as well as comparatively low-cost and uses a series of widely accepted methods, it provides researchers with easy access to the metabolites secreted by the human body. Owing to its accuracy and rapid nature, metabolomic analysis of saliva and breath (known as salivaomics and breathomics, respectively) is a rapidly growing field and has shown potential to be effective in detecting and diagnosing the early stages of numerous diseases and infections in preclinical studies. This review discusses the various collection and analyses methods currently applied in two of the least used non-invasive sample types in metabolomics, specifically their application in salivaomics and breathomics research. Some of the salient research completed in this field to date is also assessed and discussed in order to provide a basis to advocate their use and possible future scientific directions.

The synthesis and characterisation of MDMA derived from a catalytic oxidation of material isolated from black pepper reveals potential route specific impurities.

This work examines the chemical synthesis of 3,4-methylenedioxy-N-methylamphetamine (MDMA) from piperonal prepared via a catalytic ruthenium tetroxide oxidation of piperine extracted from black pepper. A variety of oxidation conditions were experimented with including different solvent systems and co-oxidants. A sample of prepared piperonal was successfully converted into MDMA via 3,4-methylenedioxyphenyl-2-nitropropene (MDP2NP) and 3,4-methylenedioxyphenyl-2-propanone (MDP2P) and the impurities within each product characterised by GC-MS to give a contaminant profile of the synthetic pathway. Interestingly, it was discovered that a chlorinated analogue of piperonal (6-chloropiperonal) was created during the oxidation process by an as yet unknown mechanism. This impurity reacted alongside piperonal to give chlorinated analogues of each precursor, ultimately yielding 2-chloro-4,5-methylenedioxymethamphetamine (6-Cl-MDMA) as an impurity within the MDMA sample. The methodology developed is a simple way to synthesise a substantial amount of precursor material with easy to obtain reagents. The results also show that chlorinated MDMA analogues, previously thought to be deliberately included adulterants, may in fact be route specific impurities with potential application in determining the origin and synthesis method of seized illicit drugs.

Screening of cannabinoids in industrial-grade hemp using two-dimensional liquid chromatography coupled with acidic potassium permanganate chemiluminescence detection.

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two-dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 Å CN column and a Poroshell 120 EC-C18 column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.

A comparative study of programs to predict direct photolysis rates in wastewater systems.

A wide range of contaminants of emerging concern (CECs) are known to photodegrade in the surface layers of natural waters and wastewater systems. Computer programs such as GCSolar, ABIWAS, APEX, EXAMS and WASP model the direct photolysis rates and half-lives of CECs, usually as a function of the solar irradiance, water molar light extinction, chemical molar light absorption and reaction quantum yield. These programs have been used extensively for studies in natural water systems in the northern hemisphere. However, their applicability to wastewater treatment systems such as waste stabilisation ponds and/or southern hemisphere conditions is not well studied. Here we present a comparative review of the major software used and their potential applicability to predicting direct photolysis rates and half-lives in wastewater. The newer equivalent monochromatic wavelength, approach, which enables the approximation of polychromatic photodegradation via a monochromatic wavelength is also discussed. Current software appears to be less suitable for modelling photodegradation in wastewater systems in the southern hemisphere than the northern hemisphere as their internal databases are based on data from natural waters in the northern hemisphere. This may be because there have been few attempts to model CEC photolysis in wastewater systems, particularly in the southern hemisphere. This indicates that either new software needs to be developed, or these programs need to be updated with data on wastewater matrices and/or the southern hemisphere. We anticipate this review will promote the adaptation of these programs as tools to further the understanding CEC photodegradation in wastewater treatment plants.

Exposure to PFAS contaminated urban wetland water causes similar metabolic alterations to laboratory-based exposures in the freshwater amphipod Austrochiltonia subtenuis.

Assessing the harm caused by pollutants in urban ecosystems remains a significant challenge. Traditional ecotoxicological endpoints are often not sensitive enough to detect the effects of toxicants at environmentally relevant concentrations (≤ng/L). A potential solution is using molecular biology methods to look at small biochemical changes caused by exposure to ng/L concentrations of contaminants. This has been tested in the lab but not conclusively demonstrated in the field. We exposed the freshwater amphipod (Austrochiltonia subtenuis) to water from an urban wetland containing known concentrations of per-and polyfluoroalkyl substances (as well as very low concentrations of pesticides) for 14 days and analyzed their metabolite profiles. Mannose, Myo-inositol, and Isopropyl propionate were found to change in PFAS exposed amphipods, a similar response to that previously observed in laboratory exposures to the same PFAS, but not pesticides. The results give a better understanding of PFAS toxicity at environmentally relevant concentrations and conditions.

Copper toxicity in the microalga Chlamydomonas reinhardtii: an integrated approach.

The effects of copper exposure at five different concentrations on the freshwater alga Chlamydomonas reinhardtii were studied at the biochemical (metabolite), physiological (uptake kinetics and flow cytometry) and growth level. Changes at the physiological level were evident at the lowest exposure concentration while effects on the metabolome and on growth only occurred at the highest copper concentration tested. Flow cytometry revealed the presence of higher reactive oxygen species concentrations in algae exposed to higher copper concentrations and this was confirmed by a significant reduction in glutathione levels as part of the metabolomics assessment. Cu(2+) uptake kinetic data contributed information on possible mechanisms of copper toxicity, revealing that, a decrease in efflux pumping might be at the basis of an increased metal accumulation at higher exposure levels. This study demonstrates the value of using a comparative approach to investigating the mechanisms of toxicity rather than focusing on a single level of organization or effect.

Steroid oestrogens in the environment: an Australian perspective.

Endocrine disrupting compounds (EDCs) have been in the scientific spotlight since the 1980s. However, there has been much less research reported in Australia than in other developed countries and little information is known about how these compounds interact with native Australian species compared to European and North American fauna. This is of concern because Australia has distinct wildlife and environments that face increasing intensity and frequency of extreme, climatic events compared to northern hemisphere countries. Since oestrogenic compounds cannot be prevented from entering wastewater their management and removal must occur at wastewater treatment plants. Biological treatment is the most effective tool in this regard; however the financial and environmental costs must be balanced with the environmental benefit to effectively plan treatment options. Since standard risk assessment models and procedures developed internationally are unlikely to translate well to Australian ecosystems, new, novel and localised research on both the monitoring and assessment of EDCs in Australian wastewater and receiving aquatic environments is recommended. This includes the development of relevant bioassays and application of treatment technologies that reflect the local community and climate.