Search

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

The application of biocatalysis for the synthesis of glycans and glycoconjugates is a well-established and successful strategy, both for small and large scale synthesis. Compared to chemical synthesis, is has the advantage of high selectivity, but biocatalysis had been largely limited to natural glycans both in terms of reactivity and substrates. This review describes recent advances in exploiting enzyme promiscuity to expand the range of substrates and reactions that carbohydrate active enzymes (CAZymes) can catalyse. The main focus is on formation and hydrolysis of glycosidic linkages, including sugar kinases, reactions that are central to glycobiotechnology. In addition, biocatalysts that generate sugar analogues and modify carbohydrates, such as oxidases, transaminases and acylases are reviewed. As carbohydrate active enzymes become more accessible and protein engineering strategies become faster, the application of biocatalysis in the generation of a wide range of glycoconjugates, beyond natural structures is expected to expand., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial β1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-β-LacNAc from lactose using β1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M 1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Aims: Emergency front of neck access (FONA) is the final step in a Can't Intubate-Can't Oxygenate (CICO) scenario. In view of maintaining simplicity and promoting standardized training, the 2015 Difficult Airway Society guidelines recommend surgical cricothyroidotomy using scalpel, bougie, and tube (SBT) as the preferred technique., Material and Methods: We undertook a survey over a 2-week period to evaluate the knowledge and training, preferred rescue technique, and confidence in performing the SBT technique. Data were collected from both anesthetists and surgeons., Results: One hundred and eighty-nine responses were collected across four hospitals in the United Kingdom. The majority of participants were anesthetists (55%). One hundred and eleven (59%) respondents were aware of the national guidelines (96.2% among anesthetists and 12.9% among surgeons). Only 71 (37.6%) respondents indicated that they had formal FONA training within the last one year. Seventy-five anesthetists (72.8%) knew that SBT equipment was readily available in their department, while most surgeons (81.2%) did not know what equipment available. One hundred and five (55.5%) respondents were confident in performing surgical cricothyroidotomy in a situation where the membrane was palpable and only in 33 (17.5%) where the cricothyroid membrane was not palpable., Conclusion: This survey has demonstrated that despite evidence of good training for anesthetists in FONA, there are still shortfalls in the training and knowledge of our surgical colleagues. In an emergency, surgeons may be required to assist or secure an airway in a CICO situation. Regular multidisciplinary training of all clinicians working with anesthetized patients should be encouraged and supported., Competing Interests: There are no conflicts of interest.

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

Little is known about the effectiveness of prevention and early intervention programs for young people and adolescents once they leave or dropout from school. The effectiveness of 18 anxiety and 26 depression studies addressing prevention in community programs were identified using systematic review methodology. Anxiety and depression symptoms were reduced in ~60% of the programs. Cognitive behavioral therapy programs were more common than other interventions and were consistently found to lower symptoms or prevent depression or anxiety. Automated or computerized interventions showed promise, with 60% of anxiety programs and 83% of depression programs yielding successful outcomes on at least one measure. Further research is needed to determine the active components of successful programs, to explore cost-effectiveness and scalability factors, to investigate individual predictors of successful outcome, and to design best practice prevention programs.