Probing the expression and adhesion of glycans involved in Helicobacter pylori infection.

Helicobacter pylori infects approximately half the human population and has an unusual infective niche of the human stomach. Helicobacter pylori is a major cause of gastritis and has been classified as a group 1 carcinogen by the WHO. Treatment involves triple or quadruple antibiotic therapy, but antibiotic resistance is becoming increasingly prevalent. Helicobacter pylori expresses certain blood group related antigens (Lewis system) as a part of its lipopolysaccharide (LPS), which is thought to assist in immune evasion. Additionally, H. pylori LPS participates in adhesion to host cells alongside several adhesion proteins. This study profiled the carbohydrates of H. pylori reference strains (SS1 and 26695) using monoclonal antibodies (mAbs) and lectins, identifying interactions between two carbohydrate-targeting mAbs and multiple lectins. Atomic force microscopy (AFM) scans were used to probe lectin and antibody interactions with the bacterial surfaces. The selected mAb and lectins displayed an increased adhesive force over the surface of the curved H. pylori rods. Furthermore, this study demonstrates the ability of anti-carbohydrate antibodies to reduce the adhesion of H. pylori 26695 to human gastric adenocarcinoma cells via AFM. Targeting bacterial carbohydrates to disrupt crucial adhesion and immune evasion mechanisms represents a promising strategy for combating H. pylori infection.

Densified collagen tubular grafts for human tissue replacement and disease modelling applications.

There is a significant need across multiple indications for an off-the-shelf bioengineered tubular graft which fulfils the mechanical and biological requirements for implantation and function but does not necessarily require cells for manufacture or deployment. Herein, we present a tissue-like tubular construct using a cell-free, materials-based method of manufacture, utilizing densified collagen hydrogel. Our tubular grafts are seamless, mechanically strong, customizable in terms of lumen diameter and wall thickness, and display a uniform fibril density across the wall thickness and along the tube length. While the method enables acellular grafts to be generated rapidly, inexpensively, and to a wide range of specifications, the cell-compatible densification process also enables a high density of cells to be incorporated uniformly into the walls of the tubes, which we show can be maintained under perfusion culture. Additionally, the method enables tubes consisting of distinct cell domains with cellular configurations at the boundaries which may be useful for modelling aortic disease. Further, we demonstrate additional steps which allow for luminal surface patterning. These results highlight the universality of this approach and its potential for developing the next generation of bioengineered grafts.

Draft genome of the bluefin tuna blood fluke, Cardicola forsteri.

The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C. forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 > 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion (> 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover > 6%. Gene prediction identified 8,564 hypothetical polypeptides, > 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts.

VIVID: A Web Application for Variant Interpretation and Visualization in Multi-dimensional Analyses.

Large-scale comparative genomics- and population genetic studies generate enormous amounts of polymorphism data in the form of DNA variants. Ultimately, the goal of many of these studies is to associate genetic variants to phenotypes or fitness. We introduce VIVID, an interactive, user-friendly web application that integrates a wide range of approaches for encoding genotypic to phenotypic information in any organism or disease, from an individual or population, in three-dimensional (3D) space. It allows mutation mapping and annotation, calculation of interactions and conservation scores, prediction of harmful effects, analysis of diversity and selection, and 3D visualization of genotypic information encoded in Variant Call Format on AlphaFold2 protein models. VIVID enables the rapid assessment of genes of interest in the study of adaptive evolution and the genetic load, and it helps prioritizing targets for experimental validation. We demonstrate the utility of VIVID by exploring the evolutionary genetics of the parasitic protist Plasmodium falciparum, revealing geographic variation in the signature of balancing selection in potential targets of functional antibodies.

Helicobacter pylori and the Role of Lipopolysaccharide Variation in Innate Immune Evasion.

Helicobacter pylori is an important human pathogen that infects half the human population and can lead to significant clinical outcomes such as acute and chronic gastritis, duodenal ulcer, and gastric adenocarcinoma. To establish infection, H. pylori employs several mechanisms to overcome the innate and adaptive immune systems. H. pylori can modulate interleukin (IL) secretion and innate immune cell function by the action of several virulence factors such as VacA, CagA and the type IV secretion system. Additionally, H. pylori can modulate local dendritic cells (DC) negatively impacting the function of these cells, reducing the secretion of immune signaling molecules, and influencing the differentiation of CD4+ T helper cells causing a bias to Th1 type cells. Furthermore, the lipopolysaccharide (LPS) of H. pylori displays a high degree of phase variation and contains human blood group carbohydrate determinants such as the Lewis system antigens, which are proposed to be involved in molecular mimicry of the host. Lastly, the H. pylori group of outer membrane proteins such as BabA play an important role in attachment and interaction with host Lewis and other carbohydrate antigens. This review examines the various mechanisms that H. pylori utilises to evade the innate immune system as well as discussing how the structure of the H. pylori LPS plays a role in immune evasion.

Global diversity and balancing selection of 23 leading Plasmodium falciparum candidate vaccine antigens.

Investigation of the diversity of malaria parasite antigens can help prioritize and validate them as vaccine candidates and identify the most common variants for inclusion in vaccine formulations. Studies of vaccine candidates of the most virulent human malaria parasite, Plasmodium falciparum, have focused on a handful of well-known antigens, while several others have never been studied. Here we examine the global diversity and population structure of leading vaccine candidate antigens of P. falciparum using the MalariaGEN Pf3K (version 5.1) resource, comprising more than 2600 genomes from 15 malaria endemic countries. A stringent variant calling pipeline was used to extract high quality antigen gene 'haplotypes' from the global dataset and a new R-package named VaxPack was used to streamline population genetic analyses. In addition, a newly developed algorithm that enables spatial averaging of selection pressure on 3D protein structures was applied to the dataset. We analysed the genes encoding 23 leading and novel candidate malaria vaccine antigens including csp, trap, eba175, ama1, rh5, and CelTOS. Our analysis shows that current malaria vaccine formulations are based on rare haplotypes and thus may have limited efficacy against natural parasite populations. High levels of diversity with evidence of balancing selection was detected for most of the erythrocytic and pre-erythrocytic antigens. Measures of natural selection were then mapped to 3D protein structures to predict targets of functional antibodies. For some antigens, geographical variation in the intensity and distribution of these signals on the 3D structure suggests adaptation to different human host or mosquito vector populations. This study provides an essential framework for the diversity of P. falciparum antigens to be considered in the design of the next generation of malaria vaccines.

Collaborative Heart Attack Management Program (CHAMP): use of prehospital thrombolytics to improve timeliness of STEMI management in British Columbia.

Coronary artery disease is the second leading cause of death in Canada. Time to treatment in ST-elevation myocardial infarction (STEMI) is directly related to morbidity and mortality. Thrombolysis is the primary treatment for STEMI in many regions of Canada because of prolonged transport times to percutaneous coronary intervention-capable centres. To reduce time from first medical contact (FMC) to thrombolysis, some emergency medical services (EMS) systems have implemented prehospital thrombolysis (PHT). PHT is not a novel concept and has a strong evidence base showing reduced mortality.Here, we describe a quality improvement initiative to decrease time from FMC to thrombolysis using PHT and aim to describe our methods and challenges during implementation. We used a quality improvement framework to collaborate with hospitals, EMS, cardiology, emergency medicine and other stakeholders during implementation. We trained advanced care paramedics to administer thrombolysis in STEMI with remote cardiologist support and aimed to achieve a guideline-recommended median FMC to needle time of <30 min in 80% of patients.Overall, we reduced our median FMC to needle time by 70%. Our baseline patients undergoing in-hospital thrombolysis had a median time of 84 min (IQR 62-116 min), while patients after implementation of PHT had a median time of 25 min (IQR 23-39 min). Patients treated within the guideline-recommended time from FMC to needle of <30 min increased from 0% at baseline to 61% with PHT. Return on investment analysis showed $2.80 saved in acute care costs for every $1.00 spent on the intervention.While we did not achieve our goal of 80% compliance with FMC to needle time of <30 min, our results show that the intervention substantially reduced the FMC to needle time and overall cost. We plan to continue with ongoing implementation of PHT through expansion to other communities in our province.

Completeness of reporting for systematic reviews of point-of-care ultrasound: a meta-research study.

OBJECTIVES: Systematic reviews are often considered among the highest quality of evidence. Completely reported systematic reviews, however, are required so readers can assess for generalisability of the research to practice and risk of bias. The objective of this study was to assess the completeness of reporting for systematic reviews assessing the diagnostic accuracy of point-of-care ultrasound (POCUS) using the Preferred Reporting Items for Systematic Reviews and Meta-analyses for Diagnostic Test Accuracy (PRISMA-DTA) checklist that was published in 2018. DESIGN AND SETTING: In this meta-research study, MEDLINE, EMBASE and Cochrane Library databases were searched, with no date restriction, on March 1st, 2020 for systematic reviews assessing the diagnostic accuracy of POCUS. Adherence to PRISMA-DTA for the main text and abstract was scored independently and in duplicate using a modified checklist. Prespecified subgroup analyses were performed. MAIN OUTCOME MEASURES: The primary outcome was the mean PRISMA-DTA checklist adherence for the full-text and abstract. RESULTS: A total of 71 studies published from 2008 to 2020 met the inclusion criteria. The overall adherence for the full-text was moderate: 19.8 out of 26.0 items (76%) and for the abstract was 7.0 out of 11.0 items (64%). Although many items in the PRISMA-DTA checklist were frequently reported, several were r infrequently reported (<33% of studies), including item 5 (protocol registration), item D2 (minimally acceptable test accuracy) and item 14 (variability in target condition, index test and reference standards). Subgroup analyses showed a higher PRISMA-DTA mean adherence (SD) for high impact journals (20.9 (2.52) vs 18.9 (1.95); p<0.001), studies including supplemental materials (20.6 (2.48) vs 18.9 (2.28); p=0.004), studies citing adherence to PRISMA reporting guidelines (20.4 (1.95) vs 19.0 (3.00); p=0.038) and studies published in journals endorsing PRISMA guidelines (20.2 (2.47) vs 18.6 (2.37); p=0.025). There was variable adherence based on journal of publication (p=0.006), but not for study population (adult vs paediatric vs mixed) (p=0.62), year of publication (p=0.94), body region (p=0.78) or country (p=0.40). There was no variability in abstract adherence based on whether the abstract was structured with subheadings or not (p=0.25). A Spearman's correlation found moderate correlation between higher word counts and abstractand full-text adherence (R=0.45, p<0.001 and R=0.38, p=0.001), respectively. CONCLUSIONS: Overall, the reporting of POCUS diagnostic accuracy systematic reviews and meta-analyses was moderate. We identified deficits in several key areas including the preregistration of systematic reviews in an online repository, handling of multiple definitions of target conditions, index tests and reference standards and specifying minimally acceptable test accuracy. Prospective registration of reviews and detailed reporting as per PRISMA-DTA during the research process could improve reporting completeness. At an editorial level, word count and supplemental material limitations may impede reporting completeness, whereas endorsement of reporting guidelines on journal websites could improve reporting.

Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

The relationship between no-flow interval and survival with favourable neurological outcome in out-of-hospital cardiac arrest: Implications for outcomes and ECPR eligibility.

BACKGROUND: The "no flow" interval is the time from out-of-hospital cardiac arrest (OHCA) to cardiopulmonary resuscitation (CPR). Its prognostic value is important to define for prehospital resuscitation decisions, post-resuscitation care and prognostication, and extracorporeal cardiopulmonary resuscitation (ECPR) candidacy assessment. METHODS: We examined bystander-witnessed OHCAs without bystander CPR from two Resuscitation Outcomes Consortium datasets. We used modified Poisson regression to model the relationship between the no-flow interval (9-1-1 call to professional resuscitation) and favourable neurological outcome (Modified Rankin Score ≤ 3) at hospital discharge. Furthermore, we identified the no-flow interval beyond which no patients had a favourable outcome. We analysed a subgroup to simulate ECPR-treated patients (witnessed arrest, age < 65, non-asystole initial rhythm, and >30 min until return of circulation). RESULTS: Of 43,593 cases, we included 7299; 616 (8.4%) had favourable neurological outcomes. Increasing no-flow interval was inversely associated with favourable neurological outcomes (adjusted relative risk 0.87, 95% CI 0.85-0.90); the adjusted probability of a favourable neurological outcome decreased by 13% (95% CI 10-15%) per minute. No patients (0/7299, 0%; 1-sided 97.5% CI 0-0.051%) had both a no-flow interval >20 min and a favourable neurological outcome. In the hypothetical ECPR group, 0/152 (0%; 1-sided 97.5% CI 0-2.4%) had both a no-flow interval >10 min and a favourable neurological outcome. CONCLUSIONS: The probability of a favourable neurological outcome in OHCA decreases by 13% for every additional minute of no-flow time until high-quality CPR, with the possibility of favourable outcomes up to 20 min.

Identifying glycan motifs using a novel subtree mining approach.

BACKGROUND: Glycans are complex sugar chains, crucial to many biological processes. By participating in binding interactions with proteins, glycans often play key roles in host-pathogen interactions. The specificities of glycan-binding proteins, such as lectins and antibodies, are governed by motifs within larger glycan structures, and improved characterisations of these determinants would aid research into human diseases. Identification of motifs has previously been approached as a frequent subtree mining problem, and we extend these approaches with a glycan notation that allows recognition of terminal motifs. RESULTS: In this work, we customised a frequent subtree mining approach by altering the glycan notation to include information on terminal connections. This allows specific identification of terminal residues as potential motifs, better capturing the complexity of glycan-binding interactions. We achieved this by including additional nodes in a graph representation of the glycan structure to indicate the presence or absence of a linkage at particular backbone carbon positions. Combining this frequent subtree mining approach with a state-of-the-art feature selection algorithm termed minimum-redundancy, maximum-relevance (mRMR), we have generated a classification pipeline that is trained on data from a glycan microarray. When applied to a set of commonly used lectins, the identified motifs were consistent with known binding determinants. Furthermore, logistic regression classifiers trained using these motifs performed well across most lectins examined, with a median AUC value of 0.89. CONCLUSIONS: We present here a new subtree mining approach for the classification of glycan binding and identification of potential binding motifs. The Carbohydrate Classification Accounting for Restricted Linkages (CCARL) method will assist in the interpretation of glycan microarray experiments and will aid in the discovery of novel binding motifs for further experimental characterisation.

3D Printable Vascular Networks Generated by Accelerated Constrained Constructive Optimization for Tissue Engineering.

One of the greatest challenges in fabricating artificial tissues and organs is the incorporation of vascular networks to support the biological requirements of the embedded cells, encouraging tissue formation and maturation. With the advent of 3D printing technology, significant progress has been made with respect to generating vascularized artificial tissues. Current algorithms to generate arterial/venous trees are computationally expensive and offer limited freedom to optimize the resulting structures. Furthermore, there is no method for algorithmic generation of vascular networks that can recapitulate the complexity of the native vasculature for more than two trees, and export directly to a 3D printing format. Here, we report such a method, using an accelerated constructive constrained optimization approach, by decomposing the process into construction, optimization, and collision resolution stages. The new approach reduces computation time to minutes at problem sizes where previous implementations have reported days. With the optimality criterion of maximizing the volume of useful tissue which could be grown around such a network, an approach of alternating stages of construction and batch optimization of all node positions is introduced and shown to yield consistently more optimal networks. The approach does not place a limit on the number of interpenetrating networks that can be constructed in a given space; indeed we demonstrate a biomimetic, liver-like tissue model. Methods to account for the limitations of 3D printing are provided, notably the minimum feature size and infill at sharp angles, through padding and angle reduction, respectively.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody.

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

Effect of Preoperative Tricuspid and/or Mitral Regurgitation on Development of Late Right-Sided Heart Failure After Insertion of the HeartWare Left Ventricular Assist Device.

Right-sided heart failure (RHF) after left ventricular assist device implantation is a significant cause of morbidity and mortality. Although multiple predictors of early RHF have been described, information on late RHF is scarce. The aim of this study was to identify predictors of late RHF in left ventricular assist device patients. A retrospective analysis of all adult patients who underwent HeartWare-ventricular assist device implantation as a bridge to transplantation in a single-centre was performed. Late RHF was defined as RHF requiring rehospitalization after 30 days of implantation. A total of 16 (10.3%) patients from 156 implantations developed late RHF. Median time to late RHF onset was 182.5 (interquartile range 105 to 618) days. Patients developing late RHF were older at surgery. A significantly higher rate of moderate or severe tricuspid regurgitation before implantation was found in patients presenting with late RHF (81.2% vs 33.5%; p <0.001). Several echocardiographic parameters at discharge postimplant, such as significant mitral regurgitation, demonstrated a strong association with late RHF. A multivariate Cox regression analysis revealed that significant preoperative tricuspid regurgitation was the strongest predictor of late RHF (hazard ratio 5.50, 95% confidence interval [1.34 to 22.58]; p = 0.02). Significant mitral regurgitation postimplantation and older age also significantly predicted late RHF. In conclusion, preoperative significant tricuspid regurgitation and mitral regurgitation after implantation predict the occurrence of late RHF.

A Blended Prehospital Ultrasound Curriculum for Critical Care Paramedics.

OBJECTIVE: Point-of-care ultrasound is a nascent and growing area of prehospital care. Most previously described ultrasound curricula for paramedics examine a single type of ultrasound scan. Here, we describe the implementation and evaluation of a prehospital ultrasound curriculum using a blended model of traditional didactics and hands-on experience with online prereading. METHODS: We recruited a prospective convenience sample of critical care paramedics without prior ultrasound experience to take part in a 2-day ultrasound course. All participants completed prereading modules built from online resources followed by a didactic review of the material and hands-on practice. Ultrasound examinations included extended focused abdominal sonography in trauma, cardiac ultrasound, thoracic ultrasound, and vascular ultrasound. A written examination evaluated ultrasound theory and image interpretation, and a practical examination evaluated image acquisition. RESULTS: Seventeen critical care paramedics completed the course with a mean grade on the written examination of 76%, with 76% of paramedics achieving the predetermined passing mark of 70% or greater. All paramedics passed the practical examination. CONCLUSION: The implementation of a prehospital critical care ultrasound program is feasible in our provincial emergency medical services system. Further assessment is necessary to determine future knowledge and skill retention as well as clinical application and utility in real-world settings.

BioStructMap: a Python tool for integration of protein structure and sequence-based features.

Summary: A sliding window analysis over a protein or genomic sequence is commonly performed, and we present a Python tool, BioStructMap, that extends this concept to three-dimensional (3D) space, allowing the application of a 3D sliding window analysis over a protein structure. BioStructMap is easily extensible, allowing the user to apply custom functions to spatially aggregated data. BioStructMap also allows mapping of underlying genomic sequences to protein structures, allowing the user to perform genetic-based analysis over spatially linked codons-this has applications when selection pressures arise at the level of protein structure. Availability and implementation: The Python BioStructMap package is available at https://github.com/andrewguy/biostructmap and released under the MIT License. An online server implementing standard functionality is available at https://biostructmap.burnet.edu.au. Supplementary information: Supplementary data are available at Bioinformatics online.