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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., (© 2023 Sharma et al.)

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.)

Background: To facilitate ablation of ventricular tachycardia (VT), an automated localization system to identify the site of origin of left ventricular activation in real time using the 12-lead ECG was developed. The objective of this study was to prospectively assess its accuracy., Methods: The automated site of origin localization system consists of 3 steps: (1) localization of ventricular segment based on population templates, (2) population-based localization within a segment, and (3) patient-specific site localization. Localization error was assessed by the distance between the known reference site and the estimated site., Results: In 19 patients undergoing 21 catheter ablation procedures of scar-related VT, site of origin localization accuracy was estimated using 552 left ventricular endocardial pacing sites pooled together and 25 VT-exit sites identified by contact mapping. For the 25 VT-exit sites, localization error of the population-based localization steps was within 10 mm. Patient-specific site localization achieved accuracy of within 3.5 mm after including up to 11 pacing (training) sites. Using 3 remotes (67.8±17.0 mm from the reference VT-exit site), and then 5 close pacing sites, resulted in localization error of 7.2±4.1 mm for the 25 identified VT-exit sites. In 2 emulated clinical procedure with 2 induced VTs, the site of origin localization system achieved accuracy within 4 mm., Conclusions: In this prospective validation study, the automated localization system achieved estimated accuracy within 10 mm and could thus provide clinical utility.

Precise structural differentiation of often isomeric glycans is important given their roles in numerous biological processes. Mass spectrometry (MS) (and tandem MS) is one of the analytical techniques at the forefront of glycan analysis given its speed, sensitivity in producing structural information as well as the fact it can be coupled to other orthogonal analytical techniques such as liquid chromatography (LC) and ion mobility spectrometry (IMS). This review describes another family of techniques that are more commonly being hybridized to MS(/MS) namely gas-phase infrared (IR) spectroscopy, whose rise is in part due to the development and improved accessibility of tunable IR lasers. Gas-phase IR can often differentiate fine isomeric differences ubiquitous within carbohydrates that MS may be 'blind' to. There are also examples of cryogenic gas-phase IR spectroscopy with much greater spectral resolution as well as hybridizing with separative methods (LC, IMS). Furthermore, collision-induced dissociation (CID) product ions can also be probed by IR, which may be beneficial to deconvolute spectra, aid analysis and build spectral libraries, thus generating novel opportunities for fragment-based approaches to analyze glycans., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Background: Endothall has been used to control submersed aquatic plants since 1960, providing broad-spectrum control of aquatic weeds. Although endothall is considered a contact herbicide, many field observations suggest that it might have systemic activity. The goals of this research were to determine endothall's (i) absorption characteristics, (ii) translocation from shoots to roots, and (iii) potential for desorption in Eurasian watermilfoil (EWM), monoecious and dioecious hydrilla., Results: Endothall absorption was linear in dioecious hydrilla up to 192 HAT, while in EWM and monoecious hydrilla absorption data best fit an asymptotic rise function. Endothall absorption in EWM, monoecious and dioecious hydrilla was 3.3, 6.6, and 11.0 times the external herbicide concentration determined by the plant concentration factor. Translocation to EWM roots reached 7.9% of total absorbed radioactivity by 192 HAT, while translocation to monoecious and dioecious hydrilla roots reached 17.8% and 16.4% by 192 HAT, respectively. For all three species, no more than 30% of absorbed endothall moved from the plant to clean water 96 HAT., Conclusion: Endothall is a very water soluble compound and yet it accumulated in these three important aquatic weeds at concentrations significantly higher than the external herbicide concentration. These data provide evidence that endothall could have systemic activity in these aquatic species. Following 14 C-endothall applications, more 14 C translocated from shoots to roots compared to the translocation of 14 C for other systemic aquatic herbicides. The final confirmation of endothall's systemic behavior requires that the radioactivity found in the root system of these aquatic plants is 14 C endothall. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

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.

Background: Cardiac rehabilitation (CR) intervention programs are currently not part of management in patients with atrial fibrillation (AF). We sought to determine the effect of CR compared with a specialized AF clinic (AFC) and usual care on outcomes in patients with AF., Methods: This was a single-centre retrospective cohort study that was carried out using 3 databases: the Hearts in Motion database (2010-2014), prospectively collected data in an AFC (2011-2014), and a retrospective chart review for patients in usual care (2009-2012). Three care pathways were compared: (1) CR; (2) AFC; and (3) usual specialist-based care. The main outcome was AF-related emergency department visits and cardiovascular hospitalizations., Results: Of 566 patients with newly diagnosed AF, 133 (23.5%) patients underwent CR, 197 patients (34.8%) attended the AFC, whereas the remaining 236 (41.7%) were followed in a usual specialist-based care clinic. At 1 year, AF-related emergency department visits and cardiovascular hospitalization rates occurred in 7.5% in the CR group, 16.8% in the AFC group, and 29.2% in usual care. After a propensity matched analysis, usual care was associated with the highest rate of the main outcome (odds ratio, 4.91; 95% confidence interval, 2.09-11.53) compared with CR, as did the AFC compared with CR (odds ratio, 2.75; 95% confidence interval, 1.14-6.6)., Conclusions: Among patients with AF, CR was associated with a lower risk of AF-related outcomes. These findings support further study of the use of CR in the management of these patients to determine the optimal model of care for AF patients., (Copyright © 2018 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Extracellular RNAs (exRNAs) can be released by numerous cell types in vitro, are often protected within vesicles, and can modify recipient cell function. To determine how the composition and cellular sources of exRNAs and the extracellular vesicles (EVs) that carry them change in vivo during tissue inflammation, we analyzed bronchoalveolar lavage fluid (BALF) from mice before and after lung allergen challenge. In the lung, extracellular microRNAs (ex-miRNAs) had a composition that was highly correlated with airway-lining epithelium. Using cell type-specific membrane tagging and single vesicle flow, we also found that 80% of detected vesicles were of epithelial origin. After the induction of allergic airway inflammation, miRNAs selectively expressed by immune cells, including miR-223 and miR-142a, increased and hematopoietic-cell-derived EVs also increased >2-fold. These data demonstrate that infiltrating immune cells release ex-miRNAs and EVs in inflamed tissues to alter the local extracellular environment., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Background: Patients with ventricular tachycardia (VT) postmyocardial infarction (MI) are a higher risk group with significant morbidity and mortality. We examined the impact of prior coronary revascularization on clinical outcomes in patients with ischemic cardiomyopathy and VT., Methods: The VANISH trial randomized 259 patients with prior MI and antiarrhythmic drug-refractory VT to receive escalated medical therapy or catheter ablation. Clinical outcomes were compared according to whether patients have undergone prior revascularization procedures. The primary outcome was a composite of death, appropriate implantable cardiac defibrillator (ICD) shock, or VT storm. The secondary outcomes included elements of the primary outcome, hospitalization, and any ventricular arrhythmia., Results: 190 patients (73%) had prior coronary revascularization. Revascularization group had more men (97% vs 83%; P  =  0.0003) and patients in that group were older (mean age 69.3 ± 7.6 vs 66.7 ± 9.2; P  =  0.04), had more renal insufficiency (22.6% vs 8.7%; P  =  0.01), and were more likely to have an implanted cardiac resynchronization device (23% vs 10%, P  =  0.03) as compared with the nonrevascularized patients. There were no significant differences in baseline medication use. There was a trend toward fewer hospitalizations in the revascularization group (64% vs 77%; P  =  0.07); there were no differences in the individual outcomes of mortality, VT storm, ICD shocks, recurrent MI, or cardiac failure., Conclusions: In this cohort of patients with an ischemic cause for VT, a history of prior coronary revascularization was not associated with a reduction in ventricular arrhythmia or mortality., (© 2018 Wiley Periodicals, Inc.)

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.

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., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

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.

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/β 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 (MS 2 ). These findings allow for integration of MS 2 with ion mobility spectrometry (IM-MS 2 ) and lead to a strategy to distinguish α- and β-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.

IR spectroscopy of gas-phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas-phase vibrational spectra in the near and mid-IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well-established DFT (Density Functionnal Theory) modeling. The proposed approach was used to elucidate the sulfate patterns in glycosaminoglycans, a ubiquitous class of mammalian carbohydrates, which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharides from heparin and chondroitin sources were resolved, highlighting the potential of infrared multiple photon dissociation spectroscopy as a novel structural tool for carbohydrates.

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.

Background: Diverse varieties of often heterogeneous glycans are ubiquitous in nature. They play critical roles in recognition events, act as energy stores and provide structural stability at both molecular and cellular levels. Technologies capable of fully elucidating the structures of glycans are far behind the other '-omic' fields. Liquid chromatography (LC) and mass spectrometry (MS) are currently the most useful techniques for high-throughput analysis of glycans. However, these techniques do not provide full unambiguous structural information and instead the gap in full sequence assignment is frequently filled by a priori knowledge of the biosynthetic pathways and the assumption that these pathways are highly conserved., Scope of the Review: This comprehensive review details the rise of the emerging analytical technique ion mobility spectrometry (IMS) (coupled to MS) to facilitate the determination of three-dimensional shape: the separation and characterization of isobaric glycans, glyco(peptides/proteins), glycolipids, glycosaminoglycans and other polysaccharides; localization of sites of glycosylation; or interpretation of the conformational change to proteins upon glycan binding., Major Conclusions: IMS is a highly promising new analytical route, able to provide rapid isomeric separation (ms timescale) of either precursor or product ions facilitating MS characterization. This additional separation also enables the deconvolution of carbohydrate MS(/MS) information from contaminating ions, improving sensitivity and reducing chemical noise. Derivation of collision cross sections (CCS) from IM-MS(/MS) data and subsequent calculations validate putative structures of carbohydrates from ab initio derived candidates. IM-MS has demonstrated that amounts of specific glycan isomers vary between disease states, which would be challenging to detect using standard analytical approaches., General Significance: IM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (© 2015 Authors; published by Portland Press Limited.)

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.

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

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.

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.

Although microbes are known to influence karst (carbonate) aquifer ecosystem-level processes, comparatively little information is available regarding the diversity of microbial activities that could influence water quality and geological modification. To assess microbial diversity in the context of aquifer geochemistry, we coupled 16S rRNA Sanger sequencing and 454 tag pyrosequencing to in situ microcosm experiments from wells that cross the transition from fresh to saline and sulfidic water in the Edwards Aquifer of central Texas, one of the largest karst aquifers in the United States. The distribution of microbial groups across the transition zone correlated with dissolved oxygen and sulfide concentration, and significant variations in community composition were explained by local carbonate geochemistry, specifically calcium concentration and alkalinity. The waters were supersaturated with respect to prevalent aquifer minerals, calcite and dolomite, but in situ microcosm experiments containing these minerals revealed significant mass loss from dissolution when colonized by microbes. Despite differences in cell density on the experimental surfaces, carbonate loss was greater from freshwater wells than saline, sulfidic wells. However, as cell density increased, which was correlated to and controlled by local geochemistry, dissolution rates decreased. Surface colonization by metabolically active cells promotes dissolution by creating local disequilibria between bulk aquifer fluids and mineral surfaces, but this also controls rates of karst aquifer modification. These results expand our understanding of microbial diversity in karst aquifers and emphasize the importance of evaluating active microbial processes that could affect carbonate weathering in the subsurface.

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.

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.

The main focus of this review is the management of active and latent tuberculosis in pregnancy in industrialized countries. The review is geared towards the healthcare professional taking care of the obstetric patient. The epidemiology of active and latent tuberculosis in the USA and recommendations for the screening of tuberculosis in pregnancy are considered. The history of treatment methodology and its relationship to the current treatment of active and latent tuberculosis in pregnancy is reviewed, and finally, a discussion of the best time to treat latent tuberculosis in a pregnant patient is undertaken, along with thoughts on future changes and advances in this field.

Currently, most cases of active tuberculosis in the United States are a result of activation of latent tuberculosis infection. In this article, the history of the epidemiology of tuberculosis and latent tuberculosis infection is reviewed. Previous and current recommendations for screening and treatment for latent tuberculosis during pregnancy and the postpartum period are discussed. A review of the literature regarding postpartum and antepartum treatment is included. Finally, the question of whether antepartum or postpartum treatment is the most beneficial is discussed.

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.

The methodology for the incorporation of azaamino-acid residues into peptides synthesised by a solid-phase method has been extended to allow azaalanine peptides to be prepared. In this way, Ac-Leu-Ser-Gly-azaAla-Gly-Phe-Ser-Leu-NH2 H-Ala-Ala-Lys-Glu-Ala-Ala-Glu-Ala -Ala-Glu-Lys-Ala-azaAla-Glu-Leu-Ala-Leu-N2H3, and H-Ala-azaAla-Lys-Glu-Ala-Ala-Glu-Ala-Ala-Glu-Lys-Ala-Ala-Glu-Leu-A la-Leu-N2H3 have been prepared. A new analogue of the Ala-Gly sequence, 3-azaalanylpropionic acid has been prepared and used to prepare the peptide analogue acetyl-Leu-Ser-Gly-azaAla-3-Prop-Phe-Ser-Leu-NH2.

Coded gestures are physical movements to which precise meanings have been attached. Their use with an electronic pointing device potentially increases the power of the pointer by allowing several distinct control functions to be incorporated. The formation of a coded gesture is invariably subject to distortions which make its automatic recognition difficult. After particular consideration of three techniques, a dynamic programming algorithm was developed and was used in the design of a real-time gesture recognizer that allows mouse-driven software to be operated purely by means of an electronic pointer. The decision was taken to implement the gesture recognizer using a widely available, obsolescent microcomputer in order to keep down costs. Despite the inevitable technical compromises in this approach, results of preliminary trials using 10 normal subjects show an average gesture recognition rate of 98.3% and a gesture confusion rate of only 0.4%.

The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.

Prostaglandins in kidney, gills, and urinary bladder of freshwater-adapted and seawater-adapted rainbow trout, Oncorhynchus mykiss (= Salmo gairdneri), and European eel, Anguilla anguilla, were determined by solid-phase extraction of tissue homogenates and high-pressure liquid chromatography. Prostaglandins E2, E1, F1 alpha, F2 alpha, and D2 and the more stable metabolite of prostacyclin, 6-keto F1 alpha, occurred in these osmoregulatory tissues. In gill filaments and kidneys of both eel and trout, prostaglandins D2 and 6-keto F1 alpha were major prostaglandins. Concentrations of these prostaglandins were significantly lower in the eel after seawater adaptation, but not in the trout. The urinary bladder of the trout contained the highest levels of prostaglandins; bladders of seawater-adapted trout contained prostaglandin D2 at 6.7 ng/mg wet tissue, the highest level of any prostaglandin determined in the present studies. Prostaglandin D2 was not detected in bladders of freshwater-adapted trout.

t-Butyloxycarbonyl-alpha-aza-(4-aminophenyl)alanine phenyl ester (III: R = NH2) has been synthesized. The rate of inhibition of trypsin (EC 3.4.21.4) by this compound (due to acylation followed by slower deacylation) shows a marked pH maximum at approximately 6. The shape of the pH-rate curve is discussed in terms of (i) the normal pH-activity curve of trypsin reacting with a charged substrate, i.e. the protonated form of the amino compound, (ii) the deprotonation of the 4-amino group with pKa 4.3, and (iii) the lower rate of reaction of the enzyme with the uncharged, deprotonated form of the ester.

Lipase (EC 3.1.1.3) from Candida cylindraceae has been immobilized by the cellulose-titanium chloride method, and on EP-400 polyethylene, with and without glutaraldehyde crosslinking, to give active preparations when assessed by their ability to catalyse the hydrolysis of tributyrin. In both cases, the use of glutaraldehyde crosslinking was shown to improve the stability of the preparations for repeated use. The lipase immobilized on EP-400 polyethylene was found to be effective in transesterification using tributyrin or triacetin as acyl donors with l-menthol as acceptor. The production of methyl butanoate and of methyl acetate using this immobilized preparation was in each case enhanced in the presence of Amberlite IR 47 Anion exchange resin (OH form).

The pH-dependence of the photo-oxidation of L-tryptophan, in the presence of Rose Bengal and Methylene Blue, has been investigated. True, initial rate constants were determined in order to circumvent errors due to secondary processes. Photo-oxidation of glycoamylase I from A. niger in the presence of Methylene Blue or Rose Bengal resulted in a pH-dependent loss of enzymic activity, which was analogous to the destruction of free L-tryptophan during photo-oxidation. The loss of enzymic activity was closely associated with the destruction of tryptophan residues in the enzyme. Significant protection of both enzymic activity and tryptophanyl residues in the enzyme molecule was achieved by performing the photo-oxidation in the presence of maltose, which is a substrate for the enzyme. The tryptophanyl residues of glucoamylase I, which had been inactivated by reaction of its carboxylic acid residues with glycine methyl ester in the presence of a water-soluble carbodi-imide, were also substantially protected by maltose. It is concluded that the active centre of glucoamylase I is a cleft lined with tryptophanyl residues that participate in the binding of the substrate. One or more carboxylic acid residues are involved in bond cleavage.

After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.

The renal and cardiovascular effects of an intravenous infusion of angiotensin II in the freshwater anaesthetised rainbow trout, Salmo gairdneri, have been reappraised, without the infusion of noradrenaline. In these fish the previously reported antidiuretic response to angiotensin II was not maintained although a systemic pressor response persisted and the tubular transport maximum for glucose remained depressed. The data suggest that angiotensin has an intrarenal action which may play a role in renal adaptation to increased environmental salinities.

The chromogenic tripeptide substrate, benzyloxycarbonylglycyl-L-prolyl-L-citrulline p-nitroanilide, is proposed for the assay of the high-alkaline proteinase, HAP-PB92, from an alkalophilic Bacillus. The assay method is sensitive, reproducible, and may be adapted for an automatic system.

The four human glycoprotein hormones FSH, LH, CG and TSH are closely related in structure, each consisting of a common alpha-subunit and a specific beta-subunit. Amino acid sequences have been established by standard methods of protein sequencing and confirmed by base sequencing of the cDNAs encoding for the polypeptide units. Speculation on the structures involved in the binding of subunits and binding to receptors has been based on examination of these sequences for common or similar regions. There are asparagine-linked carbohydrate chains attached to the subunits and in addition O-glycosidically linked carbohydrate chains in beta-CG attached to serine molecules. The carbohydrates are essential for full biological activity. The progress so far and future possibilities for the synthesis of these glycoprotein hormones by the technique of genetic engineering are discussed.