Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.

Enzyme promiscuity of carbohydrate active enzymes and their applications in biocatalysis.

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.

Advancing Solutions to the Carbohydrate Sequencing Challenge.

Carbohydrates possess a variety of distinct features with stereochemistry playing a particularly important role in distinguishing their structure and function. Monosaccharide building blocks are defined by a high density of chiral centers. Additionally, the anomericity and regiochemistry of the glycosidic linkages carry important biological information. Any carbohydrate-sequencing method needs to be precise in determining all aspects of this stereodiversity. Recently, several advances have been made in developing fast and precise analytical techniques that have the potential to address the stereochemical complexity of carbohydrates. This perspective seeks to provide an overview of some of these emerging techniques, focusing on those that are based on NMR and MS-hybridized technologies including ion mobility spectrometry and IR spectroscopy.

Role of contact force in ischemic scar-related ventricular tachycardia ablation; optimal force required and impact of left ventricular access route.

BACKGROUND: Contact force-sensing technology has become a widely used addition to catheter ablation procedures. Neither the optimal contact force required to achieve adequate lesion formation in the ventricle, nor the impact of left ventricular access route on contact force has been fully clarified. PATIENTS AND METHODS: Consecutive patients (n = 24) with ischemic cardiomyopathy who underwent ablation for scar-related ventricular tachycardia were included in the study. All ablations (n = 25) were performed using irrigated contact force-sensing catheters (Smart Touch, Biosense Webster). Effective lesion formation was defined as electrical unexcitability post ablation at sites which were electrically excitable prior to ablation (unipolar pacing at 10 mA, 2 ms pulse width). We explored the contact force which achieved effective lesion formation and the impact of left ventricular access route (retrograde aortic or transseptal) on the contact force achieved in various segments of the left ventricle. Scar zone was defined as bipolar signal amplitude < 0.5 mV. RESULTS: Among 427 ablation points, effective lesion formation was achieved at 201 points (47.1%). Contact force did not predict effective lesion formation in the overall group. However, within the scar zone, mean contact force ≥ 10 g was significantly associated with effective lesion formation [OR 3.21 (1.43, 7.19) P = 0.005]. In the 12-segment model of the left ventricle, the retrograde approach was associated with higher median contact force in the apical anterior segment (31 vs 19 g; P = 0.045) while transseptal approach had higher median force in the basal inferior segment (25 vs 15 g; P = 0.021). In the 4-segment model, the retrograde approach had higher force in the anterior wall (28 vs 16 g; P = 0.004) while the transseptal approach had higher force in the lateral wall (21 vs 18 g; P = 0.032). There was a trend towards higher force in the inferior wall with the transseptal approach, but this was not statistically significant (20 vs 15 g; P = 0.063). CONCLUSIONS: In patients with ischemic cardiomyopathy, a mean contact force of 10 g or more within the scar zone had the best correlation with electrical unexcitability post ablation in our study. The retrograde aortic approach was associated with better contact force over the anterior wall while use of a transseptal approach had better contact force over the lateral wall.

Cost Effectiveness of Ventricular Tachycardia Ablation Versus Escalation of Antiarrhythmic Drug Therapy: The VANISH Trial.

OBJECTIVES: This analysis uses the data from the randomized controlled trial to assess the cost effectiveness of catheter ablation (n = 132) versus escalated antiarrhythmic therapy (n = 127). BACKGROUND: For survivors of myocardial infarction with implantable cardioverter-defibrillator shocks despite antiarrhythmic drugs, the VANISH (Ventricular Tachycardia Ablation Versus Escalated Antiarrhythmic Drug Therapy in Ischemic Heart Disease) trial demonstrated improved clinical outcomes with catheter ablation compared with more aggressive antiarrhythmic pharmacotherapy. METHODS: Health care resource use and quality-of-life data were used to determine the cost effectiveness of catheter ablation. Published references were used to estimate costs (in 2015 Canadian dollars). The analysis was over 3 years, with a 5% discount rate. Adjustment was made for censoring and baseline utilities. RESULTS: Ablation resulted in greater quality-adjusted life-years (QALYs) than escalated drug therapy did (1.63 vs. 1.49; difference: 0.14; 95% confidence interval [CI]: -0.20 to 0.46) and higher cost ($65,126 vs. $60,269; difference: $4,857; 95% CI: -$19,757 to $27,106); with an incremental cost per QALY gained for ablation versus escalated drug therapy of $34,057 primarily due to the initial costs of ablation, which were partially offset by the costs of subsequent ablations and adverse outcomes in the escalated drug therapy arm. For patients with amiodarone-refractory ventricular tachycardia, ablation dominated escalated drug therapy, with greater QALYs (1.48 vs. 1.26; difference: 0.22; 95% CI: -0.19 to 0.59) and lower costs ($67,614 vs. $68,383; difference: -$769; 95% CI: -$35,330 to $27,092). For those with sotalol-refractory ventricular tachycardia, ablation resulted in similar QALYs (1.90 vs. 1.90; difference: -0.00; 95% CI: -0.59 to 0.62) and higher costs ($60,455 vs. $45,033; difference: $15,422; 95% CI: -$10,968 to $48,555). CONCLUSIONS: For the total trial population, results are suggestive that ablation is cost effective compared with escalation of drug therapy. This result was only manifest for the subgroup of patients whose qualifying arrhythmia occurred despite amiodarone.

Applications of a highly α2,6-selective pseudosialidase.

Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyze trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.

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.

Bottom-Up Elucidation of Glycosidic Bond Stereochemistry.

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/ß glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish α- and ß-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species.

Cyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic hydrogen cyanide (HCN) upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and high resolution MS (HR-MS) complemented by ion-mobility MS was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di- and tri-glycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and 'anitriles' derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.

The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics.

Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis.

Initial human feasibility of infusion needle catheter ablation for refractory ventricular tachycardia.

BACKGROUND: Ablation of ventricular tachycardia (VT) is sometimes unsuccessful when ablation lesions are of insufficient depth to reach arrhythmogenic substrate. We report the initial experience with the use of a catheter with an extendable/retractable irrigated needle at the tip capable of intramyocardial mapping and ablation. METHODS AND RESULTS: Sequential consenting patients with recurrent VT underwent ablation with the use of a needle-tipped catheter. At target sites, the needle was advanced 7 to 9 mm into the myocardium, permitting pacing and recording. Infusion of saline/iodinated contrast mixture excluded perforation and ensured intramyocardial deployment. Further infusion was delivered before and during temperature-controlled radiofrequency energy delivery through the needle. All 8 patients included (6 male; mean age, 54) with a mean left ventricular ejection fraction of 29% were refractory to multiple antiarrhythmic drugs, and 1 to 4 previous catheter ablation attempts (epicardial in 4) had failed. Patients had 1 to 7 (median, 2) VTs present or inducible; 2 were incessant. Some intramyocardial VT mapping was possible in 7 patients. A mean of 22 (limits, 3-48) needle ablation lesions were applied in 8 patients. All patients had at least 1 VT terminated or rendered noninducible. During a median of 12 months follow-up, 4 patients were free of recurrent VT, and 3 patients were improved, but had new VTs occur at some point during follow-up. Two died of the progression of preexisting heart failure without recurrent VT. Complications included tamponade in 1 patient and heart block in 2 patients. CONCLUSIONS: Intramyocardial infusion-needle catheter ablation is feasible and permits control of some VTs that have been refractory to conventional catheter ablation therapy, warranting further study.

Enzymatic reactions on immobilised substrates.

This review gives an overview of enzymatic reactions that have been conducted on substrates attached to solid surfaces. Such biochemical reactions have become more important with the drive to miniaturisation and automation in chemistry, biology and medicine. Technical aspects such as choice of solid surface and analytical methods are discussed and examples of enzyme reactions that have been successful on these surfaces are provided.

Dual purpose S-trityl-linkers for glycoarray fabrication on both polystyrene and gold.

There is a wide range of immobilisation reactions to tether substrates to a variety of surfaces for array-based analysis. Most of these immobilisation strategies are specific for a particular surface and require an additional linker to be attached to the substrate or the surface. Furthermore, the analysis of functionalised surfaces is often restricted to certain analytical techniques and therefore, different immobilisation strategies for different surfaces are desirable. Here we have tested an S-tritylated linker for non-covalent or covalent immobilisation of mannosides to polystyrene or gold surfaces. S-Tritylated mannosides with varying linkers were readily synthesised and used to add to biorepulsive maleimide-terminated preformed SAMs after in situ deprotection of the S-trityl group. In addition, S-tritylated mannosides themselves formed stable glycoarrays on polystyrene microtiter plates. The glycoarrays were successfully analysed by MALDI-ToF mass spectrometry, SPR spectroscopy, and interrogated with GFP-transfected Escherichia coli cells. This work has shown that a dual purpose linker can be used on multiple surfaces to form arrays allowing for different testing as well as analytical approaches.

Formation of carbohydrate-functionalised polystyrene and glass slides and their analysis by MALDI-TOF MS.

Glycans functionalised with hydrophobic trityl groups were synthesised and adsorbed onto polystyrene and glass slides in an array format. The adsorbed glycans could be analysed directly on these minimally conducting surfaces by MALDI-TOF mass spectrometry analysis after aluminium tape was attached to the underside of the slides. Furthermore, the trityl group appeared to act as an internal matrix and no additional matrix was necessary for the MS analysis. Thus, trityl groups can be used as simple hydrophobic, noncovalently linked anchors for ligands on surfaces and at the same time facilitate the in situ mass spectrometric analysis of such ligands.