Evaluation of a Novel Approach for Reducing Emissions of Pharmaceuticals to the Environment

Increased interest over the levels of pharmaceuticals detected in the environment has led to the need for new approaches to manage their emissions. Inappropriate disposal of unused and waste medicines and release from manufacturing plants are believed to be important pathways for pharmaceuticals entering the environment. In situ treatment technologies, which can be used on-site in pharmacies, hospitals, clinics, and at manufacturing plants, might provide a solution. In this study we explored the use of Pyropure, a microscale combined pyrolysis and gasification in situ treatment system for destroying pharmaceutical wastes. This involved selecting 17 pharmaceuticals, including 14 of the most thermally stable compounds currently in use and three of high environmental concern to determine the technology’s success in waste destruction. Treatment simulation studies were done on three different waste types and liquid, solid, and gaseous emissions from the process were analyzed for parent pharmaceutical and known active transformation products. Gaseous emissions were also analyzed for NOx, particulates, dioxins, furans, and metals. Results suggest that Pyropure is an effective treatment process for pharmaceutical wastes: over 99 % of each study pharmaceutical was destroyed by the system without known active transformation products being formed during the treatment process. Emissions of the other gaseous air pollutants were within acceptable levels. Future uptake of the system, or similar in situ treatment approaches, by clinics, pharmacists, and manufacturers could help to reduce the levels of pharmaceuticals in the environment and reduce the economic and environmental costs of current waste management practices

One filter, one sample and the N- and O-glyco(proteo)me: towards a system to study disorders of protein glycosylation. : Toward a System to Study Disorders of Protein Glycosylation

A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH4OH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate. Sonication achieves O-glycan release in 1 h. Combining the established protocol for filter-aided N-glycan separation, here optimized for enhanced PNGase F efficiency, with the developed O-glycan release method allows analysis of both N- and O-glycans from one sample, in the same filter unit, from 0.5 to 1 million cells. The method is compatible with subsequent analysis of the residual protein by liquid chromatography-mass spectrometry (LC-MS) after glycan release. The medium throughput approach is amenable to analysis of biological replicates, offering a simple way to assess the often subtle changes to glycan profiles accompanying differentiation and disease progression, in a statistically robust way

Detection of opium alkaloids in a Cypriot base-ring juglet

A method has been developed for extracting poppy alkaloids from oily matrices, specifically lipid residues associated with archaeological ceramics. The protocol has been applied to fresh and artificially aged poppyseed oil and to residue from a Late Bronze Age Cypriot juglet in the collections of the British Museum. The juglet is of a type that has been linked with ancient trade in opium due to its poppy-head shape and wide distribution; it is a rare example of an intact vessel with contents sealed inside. Bulk analysis of the residue by GC-EI-MS and pyGC-EI-MS indicated a degraded plant oil and possible presence of papaverine. Analysis of the alkaloid extracts by HPLC-ESI-MS using both triple quadrupole and FTICR mass spectrometers detected the five primary opium alkaloids in fresh poppyseed oil and papaverine in most of the aged samples. Papaverine and thebaine were detected in the juglet residue, providing the first rigorous chemical evidence to support a link between this vessel type and opium, or at least poppies. The association of opium with oil raises new questions about the ancient purpose of the commodities within these vessels, and the low levels (ng g−1) of opiates detected in this unusually well-preserved residue shed doubt on the scope for their detection in more fragmentary ceramic remains (potsherds). Papaverine was found to exhibit challenging carryover behaviour in all the analytical methods used in this study. The phenomenon has not been reported before and should be considered in future analyses of this analyte in all application areas

Trehalose During Two Stress Responses in Acanthamoeba : Differentiation Between Encystation and Pseudocyst Formation

The non-reducing disaccharide trehalose can serve as a protectant against a range of environmental stressors, such as heat, cold, or dehydration, in both prokaryotes and eukaryotes, with the exception of vertebrates. Here, we analyzed trehalose metabolism in the facultatively parasitic organism Acanthamoeba castellanii, known to respond to unfavorable external conditions by forming two resistant stages: a cyst, produced in the case of chronic stress, and a pseudocyst, formed in reaction to acute stress. The possible role of trehalose in the resistant stages was investigated using a combination of bioinformatic, molecular biological and biochemical approaches. Genes for enzymes from a widespread trehalose-6-synthase-trehalose-6-phosphate phosphatase (TPS-TPP) pathway and a prokaryotic trehalose synthase (TreS) pathway were identified. The expression patterns of the genes during encystation and pseudocyst formation were analyzed and correlated with the time course of cellular trehalose content determined mass spectrometrically. The data clearly demonstrate fundamental differences between encystation and pseudocyst formation at the level of cellular metabolism

Metabolomic approaches to studying the response to drought stress in corn (Zea mays) cobs

Metabolomics is a technique that allows for the evaluation of the entire extractable chemical profile of a plant, for example, using high-resolution mass spectrometry (HRMS) and can be used to evaluate plant stress responses, such as those due to drought. Metabolomic analysis is dependent upon the efficiency of the extraction protocol. Currently, there are two common extraction procedures widely used in metabolomic experiments, those that extract from plant tissue processed in liquid nitrogen or extraction from lyophilised plant tissues. Here, we evaluated the two using non-targeted metabolomics to show that lyophilisation can stabilise the maize (Zea mays) extractable metabolome, increasing throughput and efficiency of extraction as compared to the more traditional processing in liquid nitrogen. Then, we applied the lyophilisation approach to explore the effect of drought upon the maize metabolome in a non-targeted HRMS metabolomics approach. Metabolomics revealed differences in the mature maize metabolome having undergone three drought conditions imposed at two critical development stages (three-leaf stage and grain-fill stage); moreover, this difference was observed across two tissue types (kernel and inner cob/pith). It was shown that under ideal conditions, the biochemical make-up of the tissue types is different. However, under stress conditions, the stress response dominates the metabolic profile. Drought-related metabolites known from other plant systems have been identified and metabolomics has revealed potential novel drought-stress indicators in our maize system

Polyamines are required for normal growth in sinorhizobium meliloti

Polyamines (PAs) are ubiquitous polycations derived from basic L-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45% as much ODC activity and its growth rates were reduced by 42, 14 and 44% under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro

Protein sequences bound to mineral surfaces persist into deep time

Proteins persist longer in the fossil record than DNA, but the longevity, survival mechanisms and substrates remain contested. Here, we demonstrate the role of mineral binding in preserving the protein sequence in ostrich (Struthionidae) eggshell, including from the palaeontological sites of Laetoli (3.8 Ma) and Olduvai Gorge (1.3 Ma) in Tanzania. By tracking protein diagenesis back in time we find consistent patterns of preservation, demonstrating authenticity of the surviving sequences. Molecular dynamics simulations of struthiocalcin-1 and -2, the dominant proteins within the eggshell, reveal that distinct domains bind to the mineral surface. It is the domain with the strongest calculated binding energy to the calcite surface that is selectively preserved. Thermal age calculations demonstrate that the Laetoli and Olduvai peptides are 50 times older than any previously authenticated sequence (equivalent to ~16 Ma at a constant 10°C)

Pinus pinaster early hormonal defence responses to pinewood nematode (Bursaphelenchus xylophilus) infection

The pinewood nematode (PWN) is the causal agent of pine wilt disease, a pathology that affects conifer forests, mainly Pinus spp. PWN infection can induce the expression of phytohormone-related genes; however, changes at the early phytohormone level have not yet been explored. Phyto-hormones are low-abundance metabolites, and thus, difficult to quantify. Moreover, most method-ologies focus mainly on Arabidopsis or crop species. This work aimed to validate a fast (run time 6.6 min) liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) analytical method to quantify 14 phytohormones in Pinus pinaster stem tissues. This method was further applied to evaluate, for the first time, early phytohormone changes in susceptible and resistant phenotypes of P. pinaster 24, 48 and 72 h after inoculation (HAI) with PWN. A significant increase in salicylic acid (SA, 48 and 72 HAI) and jasmonic acid methyl ester (JA-ME, 72 HAI) was observed in susceptible phenotypes. Results indicate that the higher susceptibility of P. pinaster to PWN infection might result from an inefficient trigger of hypersensitive responses, with the involvement of JA and SA pathways. This work provides an important update in forest research, and adds to the current knowledge of Pinus spp. defence responses to PWN infection