Structural and functional characterisation of a stable, broad-specificity multimeric sialidase from the oral pathogen Tannerella forsythia

Sialidases are glycosyl hydrolase enzymes targeting the glycosidic bond between terminal sialic acids and underlying sugars. The NanH sialidase of Tannerella forsythia, one of the bacteria associated with severe periodontal disease plays a role in virulence. Here, we show that this broad-specificity enzyme (but higher affinity for α2,3 over α2,6 linked sialic acids) digests complex glycans but not those containing Neu5,9Ac. Furthermore, we show it to be a highly stable dimeric enzyme and present a thorough structural analysis of the native enzyme in its apo-form and in complex with a sialic acid analogue/ inhibitor (Oseltamivir). We also use non-catalytic (D237A) variant to characterise molecular interactions while in complex with the natural substrates 3- and 6-siallylactose. This dataset also reveals the NanH carbohydrate-binding module (CBM, CAZy CBM 93) has a novel fold made of antiparallel beta-strands. The catalytic domain structure contains novel features that include a non-prolyl cis-peptide and an uncommon arginine sidechain rotamer (R306) proximal to the active site. Via a mutagenesis programme, we identified key active site residues (D237, R212 and Y518) and probed the effects of mutation of residues in proximity to the glycosidic linkage within 2,3 and 2,6-linked substrates. These data revealed that mutagenesis of R306 and residues S235 and V236 adjacent to the acid–base catalyst D237 influence the linkage specificity preference of this bacterial sialidase, opening up possibilities for enzyme engineering for glycotechology applications and providing key structural information that for in silico design of specific inhibitors of this enzyme for the treatment of periodontitis

Evidence for a novel Carbohydrate Binding Module (CBM) of Tannerella forsythia NanH sialidase, key to interactions at the host-pathogen interface

Bacterial sialidases cleave terminal sialic acid from a variety of host glycoproteins, and contribute to survival and growth of many human-dwelling bacterial species, including various pathogens.Tannerella forsythia, an oral, Gram-negative, fastidious anaerobe, is a key organism in periodontal disease, and possesses a dedicated sialic acid utilisation and scavenging (nan) operon, including NanH sialidase. Here, we describe biochemical characterisation of recombinant NanH, including its action on host-relevant sialoglycans such as sialyl Lewis A and sialyl Lewis X (SLeA/X), and on human cell-attached sialic acids directly, uncovering that it is a highly active broad specificity sialidase. Furthermore, theN-terminal domain of NanH was hypothesised and proven to be capable of binding to a range of sialoglycans and non-sialylated derivatives withKdin the micromolar range, as determined by steady-state tryptophan fluorescence spectroscopy, but it has no catalytic activity in isolation from the active site. We consider this domain to represent the founding member of a novel subfamily of Carbohydrate Binding Module (CBM), involved in glycosidase-ligand binding. In addition, we created a catalytically inactive version of the NanH enzyme (FRIP→YMAP) that retained its ability to bind sialic acid-containing ligands and revealed for the first time that binding activity of a CBM is enhanced by association with the catalytic domain. Finally, we investigated the importance of Lewis-type sialoglycans onT. forsythia-host interactions, showing that nanomolar amounts of SLeA/Xwere capable of reducing invasion of oral epithelial cells byT. forsythiasuggesting that these are key ligands for bacterial-cellular interactions during periodontal disease

Preparedness for practice of newly qualified dental practitioners in the Australian context:an exploratory study

BACKGROUND: The current study explored the perspectives of preparedness for dental practice from a range of relevant stakeholders (i.e., educators, employers, final-year students, graduates, practitioners, and professional associations) using an anonymous online survey in which participants described either their preparedness for practice, or the preparedness of graduates they have encountered, across six domains. RESULTS: A total of 120 participants completed the survey. Participants were from several Australian states and territories; regional, rural, and urban locations; and working in the public and private sector. Students and new graduates generally felt prepared for activities in all the identified domains. Stakeholders reported consistently that the knowledge of dental profession graduates was at the required level to enter practice in Australia in a safe way. Activities involving the knowledge of clinical entrepreneurship and financial solvency were the dimensions where students and graduates felt least prepared (e.g., explaining fees, negotiating finances). In the domains involving clinical and technical competencies, students and new graduates self-assessed as less prepared around managing dental trauma and medical emergencies. On the other hand, activities around social and community orientation, and to a lesser extent professional attitudes and ethical judgements, were the dimensions where students and graduates felt the most prepared. CONCLUSIONS: Present findings indicate that there appear to be good standards of preparedness for practice for graduate dental professionals. This exploratory study provides insights into the nature of preparedness for Australian dental professionals and provides a basis for targeting education and professional development to address areas of need

Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host–pathogen interactions

Key to onset and progression of periodontitis is a complex relationship between oral bacteria and the host. The organisms most associated with severe periodontitis are the periodontal pathogens of the red complex: Tannerella forsythia , Treponema denticola and Porphyromonas gingivalis . These organisms express sialidases, which cleave sialic acid from host glycoproteins, and contribute to disease through various mechanisms. Here, we expressed and purified recombinant P. gingivalis sialidase SiaPG (PG_0352) and characterized its activity on a number of substrates, including host sialoglycoproteins and highlighting the inability to cleave diacetylated sialic acids – a phenomenon overcome by the NanS sialate-esterase from T. forsythia . Indeed SiaPG required NanS to maximize sialic acid harvesting from heavily O-acetylated substrates such as bovine salivary mucin, hinting at the possibility of interspecies cooperation in sialic acid release from host sources by these members of the oral microbiota. Activity of SiaPG and P. gingivalis was inhibited using the commercially available chemotherapeutic zanamivir, indicating its potential as a virulence inhibitor, which also inhibited sialic acid release from mucin, and was capable of inhibiting biofilm formation of P. gingivalis on oral glycoprotein sources. Zanamivir also inhibited attachment and invasion of oral epithelial cells by P. gingivalis and other periodontal pathogens, both in monospecies but also in multispecies infection experiments, indicating potential to suppress host–pathogen interactions of a mixed microbial community. This study broadens our understanding of the multifarious roles of bacterial sialidases in virulence, and indicates that their inhibition with chemotherapeutics could be a promising strategy for periodontitis therapy

Surgical aortic valve replacement in the era of transcatheter aortic valve implantation: a review of the UK national database

Objectives To date the reported outcomes of surgical aortic valve replacement (SAVR) are mainly in the settings of trials comparing it with evolving transcatheter aortic valve implantation. We set out to examine characteristics and outcomes in people who underwent SAVR reflecting a national cohort and therefore ‘real-world’ practice. Design Retrospective analysis of prospectively collected data of consecutive people who underwent SAVR with or without coronary artery bypass graft (CABG) surgery between April 2013 and March 2018 in the UK. This included elective, urgent and emergency operations. Participants’ demographics, preoperative risk factors, operative data, in-hospital mortality, postoperative complications and effect of the addition of CABG to SAVR were analysed. Setting 27 (90%) tertiary cardiac surgical centres in the UK submitted their data for analysis. Participants 31 277 people with AVR were identified. 19 670 (62.9%) had only SAVR and 11 607 (37.1%) had AVR+CABG. Results In-hospital mortality for isolated SAVR was 1.9% (95% CI 1.6% to 2.1%) and was 2.4% for AVR+CABG. Mortality by age category for SAVR only were: 75 years=2.2%. For SAVR+CABG these were; 2.2%, 1.8% and 3.1%. For different categories of EuroSCORE, mortality for SAVR in low risk people was 1.3%, in intermediate risk 1% and for high risk 3.9%. 74.3% of the operations were elective, 24% urgent and 1.7% emergency/salvage. The incidences of resternotomy for bleeding and stroke were 3.9% and 1.1%, respectively. Multivariable analyses provided no evidence that concomitant CABG influenced outcome. However, urgency of the operation, poor ventricular function, higher EuroSCORE and longer cross clamp and cardiopulmonary bypass times adversely affected outcomes. Conclusions Surgical SAVR±CABG has low mortality risk and a low level of complications in the UK in people of all ages and risk factors. These results should inform consideration of treatment options in people with aortic valve disease

User Centered Cognitive Maps

Two kinds of influence graphs are commonly used in artificial intelligence to modelize influence networks: bayesian networks [Naïm et al., 2004] and cognitive maps [Tolman, 1948]. Influence graphs provide mechanisms to highlight the influence between concepts. Cognitive maps represent a concept by a text and an influence by an arc to which a value is associated

Outcome following patent ductus arteriosus ligation in premature infants:A retrospective cohort analysis

BACKGROUND: The patent ductus arteriosus (PDA) is an important problem in premature infants. Surgical PDA ligation is usually only be considered when medical treatment has either failed or was contraindicated. The aims of our study were to determine the mortality and morbidity following patent ductus arteriosus ligation in premature infants, and whether prostaglandin synthetase inhibitor (PSI) use prior to ligation affects outcome. METHODS: A retrospective case note review study to determine the outcome of premature infants undergoing patent ductus arteriosus ligation in one tertiary neonatal intensive care unit and two paediatric cardiothoracic centres. RESULTS: We had follow-up data on 87 infants. Cumulative mortality rates at 7 days, 30 days and at hospital discharge were 2%, 8% and 20% respectively. The incidence of chronic lung disease, intraventricular haemorrhage, necrotising enterocolitis and retinopathy of prematurity were 77%, 39%, 26% and 28% respectively. There was no difference in mortality, incidence of chronic lung disease or duration of oxygen dependence between those who had and those who had not received a PSI prior to surgical ligation. In those who had received 2 or more courses of PSI prior to surgical ligation, there was a trend to increase in the duration of oxygen therapy and chronic lung disease, but no difference in mortality. CONCLUSION: This study shows that patent ductus arteriosus ligation is a relatively safe procedure (30 day survival 92%) but there is substantial late mortality and a high incidence of morbidity in the survivors. 2 or more courses of PSI prior to surgical ligation trends to increased oxygen dependence and chronic lung disease. This high risk population requires careful follow-up. A definitive prospective cohort study is lacking

Surgical aortic valve replacement in the era of transcatheter aortic valve implantation: a review of the UK national database.

OBJECTIVES: To date the reported outcomes of surgical aortic valve replacement (SAVR) are mainly in the settings of trials comparing it with evolving transcatheter aortic valve implantation. We set out to examine characteristics and outcomes in people who underwent SAVR reflecting a national cohort and therefore 'real-world' practice. DESIGN: Retrospective analysis of prospectively collected data of consecutive people who underwent SAVR with or without coronary artery bypass graft (CABG) surgery between April 2013 and March 2018 in the UK. This included elective, urgent and emergency operations. Participants' demographics, preoperative risk factors, operative data, in-hospital mortality, postoperative complications and effect of the addition of CABG to SAVR were analysed. SETTING: 27 (90%) tertiary cardiac surgical centres in the UK submitted their data for analysis. PARTICIPANTS: 31 277 people with AVR were identified. 19 670 (62.9%) had only SAVR and 11 607 (37.1%) had AVR+CABG. RESULTS: In-hospital mortality for isolated SAVR was 1.9% (95% CI 1.6% to 2.1%) and was 2.4% for AVR+CABG. Mortality by age category for SAVR only were: 75 years=2.2%. For SAVR+CABG these were; 2.2%, 1.8% and 3.1%. For different categories of EuroSCORE, mortality for SAVR in low risk people was 1.3%, in intermediate risk 1% and for high risk 3.9%. 74.3% of the operations were elective, 24% urgent and 1.7% emergency/salvage. The incidences of resternotomy for bleeding and stroke were 3.9% and 1.1%, respectively. Multivariable analyses provided no evidence that concomitant CABG influenced outcome. However, urgency of the operation, poor ventricular function, higher EuroSCORE and longer cross clamp and cardiopulmonary bypass times adversely affected outcomes. CONCLUSIONS: Surgical SAVR±CABG has low mortality risk and a low level of complications in the UK in people of all ages and risk factors. These results should inform consideration of treatment options in people with aortic valve disease

Oral health promotion practices of Australian community mental health professionals: a cross sectional web-based survey

BACKGROUND: This study explored the oral health promotion practices of Australian community mental health professionals working with people living with severe mental illness (SMI). METHODS: An anonymous cross-sectional web-based survey was distributed to all Community Rehabilitation and Support Workers (CRSWs) working at Neami National (n = 471), an Australian community mental health service. The validated questionnaire assessed participants' self-rated oral health knowledge and confidence (7 questions); their perceived barriers (9 questions) and attitudes (5 questions) to oral health promotion; and their oral health promotion practices (7 questions). Differences in responses between groups were analysed using Chi-square, Fisher's exact and Mann-Whitney U tests. Logistic Regression Analysis served to explore the probability of providing oral health support to mental health consumers. RESULTS: A total of 141 CRSWs were included in this study, achieving a response rate of 30 percent. Roughly two-fifths (39.0%) of participants had oral health training in the previous 12-months. The majority of CRSWs (89.3%) believed ('Agreed' or 'Strongly agreed') that mental health support workers have a role to play in promoting oral health. However, less than half (44.0%) of CRSWs practiced oral health promotion activities when working with mental health consumers. When asked about barriers to promoting oral health, 'lack of consumer interest' was the most prevalent issue. CRSWs who had oral health training were over three-times (OR 3.5, 95% CI 1.25-9.83, p = 0.017) more likely to provide oral health support. Results showed the provision of oral health support was most strongly associated with self-rated knowledge and confidence (OR 4.089, 95% CI 1.629-10.263, p = 0.003) and attitudes to oral health promotion (OR 3.906, 95% CI 1.77-8.65, p = 0.001). CONCLUSION: The results of this study suggest that mental health support workers who have more positive attitudes to oral health promotion and who have higher self-rated oral health knowledge and confidence are more likely to provide oral health support in their professional role. Training for community mental health professionals is essential to build confidence and skills to promote oral health for mental health consumers