Characterisation of novel lignocellulosic enzymes from the shipworm symbiont Teredinibacter turnerae

Biomass is a renewable resource which can be broken down into bio-derived high value molecules using industrial processing, parts of which rely on enzymatic degradation. Nature provides a vast pool of highly efficient lignocellulosic enzymes capable of breaking down the multitude of natural polymers found in biomass. Identifying organisms that thrive within ecological niches based around the consumption of lignocellulosic material is likely to yield novel biomass degrading enzymes. Shipworms are marine organisms which use shell-like protrusions to burrow into submerged wooden substrates in oceanic environments. Endosymbiotic bacteria, Teredinibacter turnerae, found within the gills of the shipworm is thought to provide the host with a variety of carbohydrate active enzymes for use in the digestion. This work characterises carbohydrate active enzymes identified within the genome of T. turnerae including glycoside hydrolases (GH) from families 5, 8 and 12 as well as a lytic polysaccharide monooxygenase (LPMO) from subfamily AA10. Analysis of three GH5 proteins found activities on cellulose (TtGH5_2 and TtGH5_un) and xyloglucan (TtGH5_4), with one protein (TtGH5_un) likely representing a new GH5 subfamily. TtGH8 was found to be highly active on xylans, with kinetics and structural studies showing a preference for longer xylooligosaccharides. The molecular structure of TtGH12 was solved, but substrate specificity remains unknown, indicating a potentially new activity subclass within the GH12 family. TtAA10 was active on crystalline cellulose through copper mediated oxidative attack at C1 or C4 either side of the glycosidic bond, and modelled structurally at high resolution. Like many other specialised organisms, the genome of T. turnerae is a treasure chest full of potentially useful lignocellulose degrading proteins. This work has assessed the activity and structure of several enzymes from T. turnerae and found multiple different substrate activities and potentially novel functions, paving the way for further experimentation and potential utilisation in industry

Perceptions and experiences of wrist surgeons on the management of triangular fibrocartilage complex tears: a qualitative study

There is lack of consensus on the management of triangular fibrocartilage injuries. The aim of this study was to investigate wrist surgeons’ experiences and perceptions regarding treatment of triangular fibrocartilage complex injuries and to explore the rationale behind clinical decision-making. A purposive sample of consultant wrist surgeons (n=10) was recruited through ‘snow-balling’ until data saturation was reached. Semi-structured interviews were conducted, digitally recorded and transcribed verbatim. Two researchers independently analysed data using an iterative/thematic approach. Findings suggest that surgeons rely more on their own training and experience, and patient-related factors such as individual expectations, rather than on published material, to inform their decision-making. Current classification systems are largely considered to be unhelpful

Triggers of Breathlessness in Inducible Laryngeal Obstruction and Asthma

BackgroundInducible laryngeal obstruction (ILO) is often misdiagnosed as, or may coexist with, asthma. Identifying differences in triggering factors may assist clinicians to differentiate between the two conditions, and could give mechanistic insights.ObjectiveTo identify and compare patient‐reported triggers in ILO and asthma.MethodsThis was a two‐part study. Initially we conducted a retrospective case note review of the triggers of ILO from endoscopically‐confirmed ILO patients to generate a Breathlessness Triggers Survey (BrTS). Triggers were categorised as: scents, environmental factors, temperature, emotions, mechanical factors and daily activities. Secondly, ILO and/or asthma patients completed the BrTS prospectively, rating the likelihood of each item triggering their symptoms using a five‐point Likert scale (strongly disagree to strongly agree). Chi‐square testing was performed to compare responses by cohort.ResultsData from 202 patients with ILO [73% female, mean (SD) age 53(16) years] were included in the case note review. For the prospective study, 38 patients with ILO‐only [63% females, age 57(16) years], 39 patients with asthma‐only [(56% female, age 53(13) years] and 12 patients with both ILO and asthma [83% female, mean age, 57 (14) years)] completed the BrTS. The triggers identified in the case note review were confirmed in the independent sample of patients with ILO and/or asthma and identified several difference in prevalence of the triggers between disease types. Mechanical factors [talking (

Application of sludge-based carbonaceous materials in a hybrid water treatment process based on adsorption and catalytic wet air oxidation

This paper describes a preliminary evaluation of the performance of carbonaceous materials prepared from sewage sludges (SBCMs) in a hybrid water treatment process based on adsorption and catalytic wet air oxidation; phenol was used as the model pollutant. Three different sewage sludges were treated by either carbonisation or steam activation, and the physico-chemical properties of the resultant carbonaceous materials (e.g. hardness, BET surface area, ash and elemental content, surface chemistry) were evaluated and compared with a commercial reference activated carbon (PICA F22). The adsorption capacity for phenol of the SBCMs was greater than suggested by their BET surface area, but less than F22; a steam activated, dewatered raw sludge (SA_DRAW) had the greatest adsorption capacity of the SBCMs in the investigated range of concentrations (<0.05 mol L−1). In batch oxidation tests, the SBCMs demonstrated catalytic behaviour arising from their substrate adsorptivity and metal content. Recycling of SA_DRAW in successive oxidations led to significant structural attrition and a hardened SA_DRAW was evaluated, but found to be unsatisfactory during the oxidation step. In a combined adsorption–oxidation sequence, both the PICA carbon and a selected SBCM showed deterioration in phenol adsorption after oxidative regeneration, but a steady state performance was reached after 2 or 3 cycles

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis

An evaluation of a pilot of daily testing of SARS-CoV-2 contacts in acute hospital and ambulance trusts in England

OBJECTIVES: Healthcare worker (HCW) SARS-CoV-2 contacts in England have been required to quarantine, creating staff shortages. We piloted daily contact testing (DCT) to assess its feasibility as an alternative. STUDY DESIGN: Observational service evaluation. METHODS: We conducted an observational service evaluation of seven-day daily contact testing using antigen lateral flow devices, (LFDs) at four acute hospital trusts and one ambulance trust in England. Mixed methods were employed, utilising aggregate and individual-level test monitoring data, semi-structured interviews, and a survey of eligible contacts. RESULTS: In total, 138 HCWs were identified as contacts of a confirmed SARS-CoV-2 case. Of these, 111 (80%) consented to daily LFD testing, of whom 82 (74%) completed the required programme without interruption, and 12 (11%) completed with interruption. Fifty-eight (52%) participants and two (7·4%) non-participants completed the survey. In total, 28 interviews were conducted with participants, site and infection control leads, and union representatives. One participant tested positive on LFD and polymerase chain reaction (PCR) test. Three participants tested positive on PCR but not LFD. DCT was well-accepted by trusts and staff. Participants reported no relaxation of their infection prevention and control behaviours. No incidents of transmission were detected. An estimated 729 potential days of work absence were averted. CONCLUSIONS: DCT can be acceptably operated in a healthcare setting, averting quarantine-related work absences in HCW SARS-CoV-2 contacts

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 alleles. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving allelic differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads - this was observed as early as day 3, the earliest that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse allele expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and identifying mosaic gene formation unprecedentedly early during the infection process