Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

A study of bacteria producing carbonic anhydrase enzyme for CaCO3 precipitation and soil biocementation.

We study the carbonic anhydrase (CA) pathway using autochthonous CA-producing bacteria as a means of inducing calcite precipitation, which acts as a biocement to improve the engineering soil properties. Forty different microbial strains producing CA were isolated from the foundation soil of a railway embankment in Prickwillow, UK. Three of the best CA-producing strains were selected and identified by DNA sequencing as Bacillus licheniformis, Bacillus toyonensis and Bacillus pumilus with CA activity values respectively of 1.79 U/ml, 1.42 U/ml and 1.55 U/ml. To optimise the treatments, we investigated the effect of pH, temperature, zinc co-factor and cementation solution molarity on the growth and CA activity and bioprecipitates, with CO2 added in the form of bicarbonate. Scanning electron microscope (SEM) analysis of the bioprecipitates showed that these had characteristic morphologies of calcite and vaterite crystals. The formation of calcite was further corroborated by FT-IR and Raman analysis of bioprecipitates. The precultured bacteria were injected into the fine-grained soil together with cementation solution. Unconfined compressive strength in treated soil increased up to 1 MPa, and its calcium carbonate content increased by 2.78%. This, as well as the stability of the treated soil upon water immersion, proved the biocementation of the fine-grained soil. These findings suggest the potential of employing the CA biocementation route for soil stabilisation pending further development of the technique.

Natural based products for cleaning copper and copper alloys artefacts.

Copper alloys objects can deteriorate their conservation state through irreversible corrosion. Since in the cultural heritage field every artefact is unique and any loss irreplaceable, solutions for conservation are needed. Hence, there is the necessity to stop the corrosion process with a suitable cleaning and conservation process to avoid further degradation processes without changing its morphological aspect. Chelating solutions are commonly used in chemical cleaning, mainly sodium salts of ethylenediaminetetraacetic acid (EDTA). However, it is resistant to water purification procedures and is not biodegradable. The goal of this study was to see if applying an ecologically friendly chelating agent as an alternative to EDTA cleaning procedures for cultural heritage was suitable. In this study were chosen six natural-based chelators that could be a new green non-toxic alternative to EDTA in corrosion-inhibiting properties. They were tested for cleaning copper artefacts exposed to atmospheric environment in polluted areas. The study considered four amino acids, a glucoheptonate (CSA) and an industrial green chelator (GLDA). The effectiveness was tested on corrosion copper compounds and on laboratory corroded copper sheets. Finally, the cleaning efficacy was tested on four Roman coins and a modern copper painting. To define the cleaning efficacy, surface analytical investigations have been carried out by means ICP-OES, UV-VIS, µ-Raman, spectro-colorimetry, XRD and FTIR. Among the amino acids, alanine was the most effective, showing an unaltered noble patina and a good effective copper recovery from corrosion patinas.

Absence of Staphylococcus aureus in Wild Populations of Fish Supports a Spillover Hypothesis.

Staphylococcus aureus is a human commensal and opportunistic pathogen that also infects other animals. In humans and livestock, where S. aureus is most studied, strains are specialized for different host species. Recent studies have also found S. aureus in diverse wild animals. However, it remains unclear whether these isolates are also specialized for their hosts or whether their presence is due to repeated spillovers from source populations. This study focuses on S. aureus in fish, testing the spillover hypothesis in two ways. First, we examined 12 S. aureus isolates obtained from the internal and external organs of a farmed fish. While all isolates were from clonal complex 45, genomic diversity indicates repeated acquisition. The presence of a φSa3 prophage containing human immune evasion genes suggests that the source was originally human. Second, we tested for S. aureus in wild fish that were isolated from likely sources. In particular, we sampled 123 brown trout and their environment at 16 sites in the remote Scottish Highlands with variable levels of exposure to humans, birds, and livestock. This screen found no S. aureus infection in any of the wild populations or their environment. Together, these results support that the presence of S. aureus in fish and aquaculture is due to spillover from humans rather than specialization. Given the trends of increasing fish consumption, a better understanding of the dynamics of S. aureus spillover in aquaculture will mitigate future risks to fish and human health. IMPORTANCE Staphylococcus aureus is a human and livestock commensal but also an important pathogen responsible for high human mortality rates and economic losses in farming. Recent studies show that S. aureus is common in wild animals, including fish. However, we do not know whether these animals are part of the normal host range of S. aureus or whether infection is due to repeated spillover events from true S. aureus hosts. Answering this question has implications for public health and conservation. We find support for the spillover hypothesis by combining genome sequencing of S. aureus isolates from farmed fish and screens for S. aureus in isolated wild populations. The results imply that fish are unlikely to be a source of novel emergent S. aureus strains but highlight the prominence of the spillover of antibiotic-resistant bacteria from humans and livestock. This may affect both future fish disease potential and the risk of human food poisoning.

Characterisation of "flushable" and "non-flushable" commercial wet wipes using microRaman, FTIR spectroscopy and fluorescence microscopy: to flush or not to flush.

The introduction to the market of wet wipes, advertised and labelled as "flushable", has been the subject of controversy due to their perceived potential to block sewer systems as observed with other non-woven cloths such as traditional non-flushable wipes. Non-woven cloths that enter wastewater systems can find their way into the aquatic environment via wastewater effluents and it has been suggested that the breakdown of these fabrics can release materials such as microplastics into the environment. Worldwide research has revealed the alarming number of aquatic organisms affected by the presence of plastic debris in the aquatic environment harbouring a potential risk to humans through the introduction of microplastics into the food chains. However, the actual material composition of flushable wipes, their fate and impacts in the aquatic environment have not yet been scientifically studied. This paper investigates the fibre composition of flushable and non-flushable wipes, specifically with regard to synthetic polymer material, using Fourier transform infrared (FTIR) and microRaman spectroscopy along with fluorescence microscopy. The study demonstrated the presence of polyester (polyethylene terephthalate, (PET)), high-density polyethylene (HDPE) and polyethylene/vinyl acetate (PEVA/EVA) in some flushable wipes and PET in all non-flushable. Other polymers such us polypropylene (PP), low-density polyethylene (LDPE), expanded polystyrene (EPS) and polyurethane (PU) were also identified as potential components in the flushable material. Hence, commercially available wet wipes labelled as flushable could also be considered as a possible source of microplastic fibres in the wastewater streams and, if not retained, in the environment.

Consideration of the bioavailability of metal/metalloid species in freshwaters: experiences regarding the implementation of biotic ligand model-based approaches in risk assessment frameworks.

After the scientific development of biotic ligand models (BLMs) in recent decades, these models are now considered suitable for implementation in regulatory risk assessment of metals in freshwater bodies. The BLM approach has been described in many peer-reviewed publications, and the original complex BLMs have been applied in prospective risk assessment reports for metals and metal compounds. BLMs are now also recommended as suitable concepts for the site-specific evaluation of monitoring data in the context of the European Water Framework Directive. However, the use is hampered by the data requirements for the original BLMs (about 10 water parameters). Recently, several user-friendly BLM-based bioavailability software tools for assessing the aquatic toxicity of relevant metals (mainly copper, nickel, and zinc) became available. These tools only need a basic set of commonly determined water parameters as input (i.e., pH, hardness, dissolved organic matter, and dissolved metal concentration). Such tools seem appropriate to foster the implementation of routine site-specific water quality assessments. This work aims to review the existing bioavailability-based regulatory approaches and the application of available BLM-based bioavailability tools for this purpose. Advantages and possible drawbacks of these tools (e.g., feasibility, boundaries of validity) are discussed, and recommendations for further implementation are given.