Defining inflammatory cell states in rheumatoid arthritis joint synovial tissues by integrating single-cell transcriptomics and mass cytometry.

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

Outcomes from a national screening program for Ukrainian refugees at risk of drug resistant tuberculosis in Wales.

High rates of drug-resistant tuberculosis in Ukraine suggest screening is necessary to mitigate public health hazards for host populations. A pathway was implemented in Wales and data prospectively collected Between 8 April and 21 December 2022. Of 5425 Ukrainian arrivals, notifications were received by TB teams on 2395 (44%) of whom 1955 (82%) were screened. The refugees were young (median age 30, IQR 14-41), and predominantly female (66.1%). Interferon- gamma release assay (IGRA) tests were positive in 112 (6.5%). One Case of active tuberculosis was identified (0.05%). Our data supports European guidelines that routine screening of this population is not recommended, but we remain uncertain as to the risks of this population going forwards.

Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial.

BACKGROUND: Although targeted biological treatments have transformed the outlook for patients with rheumatoid arthritis, 40% of patients show poor clinical response, which is mechanistically still unexplained. Because more than 50% of patients with rheumatoid arthritis have low or absent CD20 B cells-the target for rituximab-in the main disease tissue (joint synovium), we hypothesised that, in these patients, the IL-6 receptor inhibitor tocilizumab would be more effective. The aim of this trial was to compare the effect of tocilizumab with rituximab in patients with rheumatoid arthritis who had an inadequate response to anti-tumour necrosis factor (TNF) stratified for synovial B-cell status. METHODS: This study was a 48-week, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial (rituximab vs tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis; R4RA) done in 19 centres across five European countries (the UK, Belgium, Italy, Portugal, and Spain). Patients aged 18 years or older who fulfilled the 2010 American College of Rheumatology and European League Against Rheumatism classification criteria for rheumatoid arthritis and were eligible for treatment with rituximab therapy according to UK National Institute for Health and Care Excellence guidelines were eligible for inclusion in the trial. To inform balanced stratification, following a baseline synovial biopsy, patients were classified histologically as B-cell poor or rich. Patients were then randomly assigned (1:1) centrally in block sizes of six and four to receive two 1000 mg rituximab infusions at an interval of 2 weeks (rituximab group) or 8 mg/kg tocilizumab infusions at 4-week intervals (tocilizumab group). To enhance the accuracy of the stratification of B-cell poor and B-cell rich patients, baseline synovial biopsies from all participants were subjected to RNA sequencing and reclassified by B-cell molecular signature. The study was powered to test the superiority of tocilizumab over rituximab in the B-cell poor population at 16 weeks. The primary endpoint was defined as a 50% improvement in Clinical Disease Activity Index (CDAI50%) from baseline. The trial is registered on the ISRCTN database, ISRCTN97443826, and EudraCT, 2012-002535-28. FINDINGS: Between Feb 28, 2013, and Jan 17, 2019, 164 patients were classified histologically and were randomly assigned to the rituximab group (83 [51%]) or the tocilizumab group (81 [49%]). In patients histologically classified as B-cell poor, there was no statistically significant difference in CDAI50% between the rituximab group (17 [45%] of 38 patients) and the tocilizumab group (23 [56%] of 41 patients; difference 11% [95% CI -11 to 33], p=0·31). However, in the synovial biopsies classified as B-cell poor with RNA sequencing the tocilizumab group had a significantly higher response rate compared with the rituximab group for CDAI50% (rituximab group 12 [36%] of 33 patients vs tocilizumab group 20 [63%] of 32 patients; difference 26% [2 to 50], p=0·035). Occurrence of adverse events (rituximab group 76 [70%] of 108 patients vs tocilizumab group 94 [80%] of 117 patients; difference 10% [-1 to 21) and serious adverse events (rituximab group 8 [7%] of 108 vs tocilizumab group 12 [10%] of 117; difference 3% [-5 to 10]) were not significantly different between treatment groups. INTERPRETATION: The results suggest that RNA sequencing-based stratification of rheumatoid arthritis synovial tissue showed stronger associations with clinical responses compared with histopathological classification. Additionally, for patients with low or absent B-cell lineage expression signature in synovial tissue tocilizumab is more effective than rituximab. Replication of the results and validation of the RNA sequencing-based classification in independent cohorts is required before making treatment recommendations for clinical practice. FUNDING: Efficacy and Mechanism Evaluation programme from the UK National Institute for Health Research.

Microbial communities of halite deposits and other hypersaline environments.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. While the microbiology of ancient evaporites has been well studied, the ecology of halite deposits and more recently formed NaCl "salticle" stalactite structures (speleothems) in a Triassic halite mine are less well characterized. The microbiome of Kilroot Salt Mine was profiled using conventional and enhanced culturing techniques. From this, 89 halophilic archaeal isolates from six known genera, and 55 halophilic or halotolerant bacterial isolates from 18 genera were obtained. Culture-independent metagenomic approaches also revealed that culturing techniques were inadvertently biased toward specific taxa, and the need for optimized isolation procedures are required to enhance cultivation diversity. Speleothems formed from saturated brines are unique structures that have the potential to entomb haloarchaea cells for thousands of years within fluid inclusions. The presence of such fluid inclusions, alongside the high abundance of genes related to glycerol metabolism, biofilm formation, and persister cell formation is highly suggestive of an environmental niche that could promote longevity and survivability. Finally, previous studies reporting the discovery of novel biocatalysts from the Kilroot mine microbiome, suggests that this environment may be an untapped source of chemical diversity with high biodiscovery potential.

Phases of Match-Play in Professional Australian Football: Positional Demands and Match-Related Fatigue.

This study examined the influence of player position and match quarter on activity profiles during the phases of play in Australian Football. Global positioning satellite data was collected for one season from an Australian Football League team for nomadic, key position and ruck players (age: 24.8 ± 4.2 years, body mass: 88.3 ± 8.7 kg, height: 1.88 ± 0.8 m). Separate linear mixed models and effect sizes were used to analyse differences between positions and game quarter within each phase of play for values of distance, speed and metabolic power indices. There were clear differences between positions for low-speed running, high-speed running, total distance and average speed. Nomadic players generally recorded the highest match running outputs, followed by key position players and ruckmen. Within each position, offence and defence involved the highest intensities, followed by contested play and then stoppage periods. Across the four quarters, there were small to large reductions in average speed, high-speed running, high power and energy expenditure during offence, defence and contested play, but not during stoppages. Accordingly, conditioning staff should consider the intermittent intensities of the phases of match-play for each position to optimally prepare players for competition. Reductions in match intensities were evident during active periods of play providing implications for real-time monitoring to optimise the timing of rotations.

Microbiology of a NaCl stalactite 'salticle' in Triassic halite.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less-well understood. Here, the microbiology of a NaCl stalactite ('salticle') in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine-pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine-pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress-protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt-saturated habitats that are protected from ionizing radiation and have a stable water activity.

IL-23 skin and joint profiling in psoriatic arthritis: novel perspectives in understanding clinical responses to IL-23 inhibitors.

OBJECTIVES: To determine the relationship between synovial versus skin transcriptional/histological profiles in patients with active psoriatic arthritis (PsA) and explore mechanistic links between diseased tissue pathology and clinical outcomes. METHODS: Twenty-seven active PsA patients were enrolled in an observational/open-label study and underwent biopsies of synovium and paired lesional/non-lesional skin before starting anti-tumour necrosis factor (TNF) (if biologic-naïve) or ustekinumab (if anti-TNF inadequate responders). Molecular analysis of 80-inflammation-related genes and protein levels for interleukin (IL)-23p40/IL-23p19/IL-23R were assessed by real-time-PCR and immunohistochemistry, respectively. RESULTS: At baseline, all patients had persistent active disease as per inclusion criteria. At primary end-point (16-weeks post-treatment), skin responses favoured ustekinumab, while joint responses favoured anti-TNF therapies. Principal component analysis revealed distinct clustering of synovial tissue gene expression away from the matched skin. While IL12B, IL23A and IL23R were homogeneously expressed in lesional skin, their expression was extremely heterogeneous in paired synovial tissues. Here, IL-23 transcriptomic/protein expression was strongly linked to patients with high-grade synovitis who, however, were not distinguishable by conventional clinimetric measures. CONCLUSIONS: PsA synovial tissue shows a heterogeneous IL-23 axis profile when compared with matched skin. Synovial molecular pathology may help to identify among clinically indistinguishable patients those with a greater probability of responding to IL-23 inhibitors.

n-Butyllithium-Induced Tandem [3,3]-Sigmatropic Rearrangement and Carbonyl Olefination of Allyl-1,1-dichlorovinyl Ethers.

Exposure of dichlorovinyl ethers 1 to n-butyllithium and addition of saturated or unsaturated aldehydes, ketones, or esters at ambient temperature furnishes rearranged α,ß-unsaturated carboxylic acids, isolated as their corresponding methyl esters 2 in 48-91% overall yields. Exposure of dichlorovinyl ethers 1 to n-butyllithium, addition of aldehydes, ketones, dialdehydes, or diketones at -78 °C, and warming to 80 °C in the presence of SiO2 provide 1,4-dienes 3 or cycloalken-1-ols (or their dehydration products) 4 in 45-72% overall yields.

Electrochemical quantification of d-glucose during the production of bioethanol from thermo-mechanically pre-treated wheat straw.

Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10-0.13 g/g (dry weight) of d-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of d-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06-0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08-0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition.

Regular Solution Theory for Polymer Permeation Transients: A Toolkit for Understanding Experimental Waveshapes.

The accurate measurement of permeation is important at the product design stage for a variety of industries as diverse as conveyance methods for oil and gas produced fluids, such as mixtures of carbon dioxide, methane, hydrogen sulfide, water, and hydrocarbons, and in polymer-lined, unbonded flexible risers and flow lines through connectors and valves, hydrogen and methane gas carrying domestic lines, hydrogen storage tanks, sulfur hexafluoride circuit breakers for high power-carrying lines, oxygen through display technology, and drug delivery. It would also be appropriate to monitor the permeation rate through the polymer, composite, and elastomeric layers during the in-service times where applications allow. In the future, any alteration in the short term and long-term transport rates could be analyzed in terms of an initial alteration or degradation of the polymeric materials and, in some cases, metallic components. Crucially, such measurements would serve as an early warning system of any change in a polymeric material that could result in the loss of function of the fluid of a gas containing barrier material. Most experimental determinations are made through recording flux transients (varying flux) through permeation cells in which a polymer membrane or film separates a donor compartment (usually an infinite supply) and an acceptor compartment and in which membrane transport is considered to be slow. Treatment of the resulting experimental data is usually, but not always, undertaken through comparison with a steady-state model based on Fickian diffusion through the membrane, so as to extract the membrane permeability, the diffusion coefficient of the permeant, and the solubility of the permeant in the membrane phase. However, in spite of these measurements being undertaken routinely using closed cell manometric or continuous flow methods, there is a lack of literature in which experimental flux transients are provided, and in several cases, it is clear that the experimental data do not conform to the expected model of slow, Fickian diffusion through the membrane, even though experiments are performed at temperatures much higher than the glass transition temperature of the polymer membrane. In this paper, we first re-examine the classical model for an infinite source and extend it to account for (1) molecular interactions between membrane and permeant, using regular solution theory, (2) slow transport in the acceptor phase, and (3) slow kinetics across the membrane|acceptor interface. We demonstrate that all three aspects can cause permeation flux transients to exhibit unusual, nonclassical waveshapes, which have nevertheless been experimentally realized without rationalization. This enables the development of an algorithmic toolkit for the interpretation of permeation flux transients, so as to provide reliable and accurate data analysis for experimentalists.

Application of ω-Transaminases in the Pharmaceutical Industry.

Chiral amines are valuable building blocks for the pharmaceutical industry. ω-TAms have emerged as an exciting option for their synthesis, offering a potential "green alternative" to overcome the drawbacks associated with conventional chemical methods. In this review, we explore the application of ω-TAms for pharmaceutical production. We discuss the diverse array of reactions available involving ω-TAms and process considerations of their use in both kinetic resolution and asymmetric synthesis. With the aid of specific drug intermediates and APIs, we chart the development of ω-TAms using protein engineering and their contribution to elegant one-pot cascades with other enzymes, including carbonyl reductases (CREDs), hydrolases and monoamine oxidases (MAOs), providing a comprehensive overview of their uses, beginning with initial applications through to the present day.

Transaminases for industrial biocatalysis: novel enzyme discovery.

Transaminases (TAms) are important enzymes for the production of chiral amines for the pharmaceutical and fine chemical industries. Novel TAms for use in these industries have been discovered using a range of approaches, including activity-guided methods and homologous sequence searches from cultured microorganisms to searches using key motifs and metagenomic mining of environmental DNA libraries. This mini-review focuses on the methods used for TAm discovery over the past two decades, analyzing the changing trends in the field and highlighting the advantages and drawbacks of the respective approaches used. This review will also discuss the role of protein engineering in the development of novel TAms and explore possible directions for future TAm discovery for application in industrial biocatalysis. KEY POINTS: • The past two decades of TAm enzyme discovery approaches are explored. • TAm sequences are phylogenetically analyzed and compared to other discovery methods. • Benefits and drawbacks of discovery approaches for novel biocatalysts are discussed. • The role of protein engineering and future discovery directions is highlighted.

Phases of match-play in professional Australian Football: Distribution of physical and technical performance.

The current study aimed to describe the distribution of physical and technical performance during the different phases of play in professional Australian Football. The phases of play (offence, defence, contested play, umpire stoppages, set shots and goal resets) were manually coded from video footage for a single team competing in 18 matches in the Australian Football League. Measures of physical performance including total distance (m), average speed (m · min-1), low-speed running (LSR, <14.4 km h-1), high-speed running (HSR, >14.4 km h-1), accelerations (2.78 m · s-2) and decelerations (-2.78 m · s-2) were derived from each phase of play via global positioning system (GPS) devices. Technical skill data including tackles, handballs and kicks were obtained from a commercial statistics provider and derived from each phase of play. Linear mixed-effects models and effect sizes were used to assess and reflect the differences in physical and technical performance between the six phases of play. Activity and recovery cycles, defined as periods where the ball was in or out of play were also described using mean and 95% confidence intervals. The analysis showed that several similarities existed between offence and defence for physical performance metrics. Contested play involved the highest total distance, LSR, accelerations, decelerations and tackles compared to all other phases. Offence and defence involved the highest average speed and HSR running distances. Handballs and kicks were highest during offence, while tackles were highest during contested play, followed by defence. Activity and recovery cycles involved mean durations of ~110 and ~39 s and average speeds of ~160 and ~84 m · min-1, respectively. The integration of video, GPS and technical skill data can be used to investigate specific phases of Australian Football match-play and subsequently guide match analysis and training design.

Synovial tissue signatures enhance clinical classification and prognostic/treatment response algorithms in early inflammatory arthritis and predict requirement for subsequent biological therapy: results from the pathobiology of early arthritis cohort (PEAC).

OBJECTIVE: To establish whether synovial pathobiology improves current clinical classification and prognostic algorithms in early inflammatory arthritis and identify predictors of subsequent biological therapy requirement. METHODS: 200 treatment-naïve patients with early arthritis were classified as fulfilling RA1987 American College of Rheumatology (ACR) criteria (RA1987) or as undifferentiated arthritis (UA) and patients with UA further classified into those fulfilling RA2010 ACR/European League Against Rheumatism (EULAR) criteria. Treatment requirements at 12 months (Conventional Synthetic Disease Modifying Antirheumatic Drugs (csDMARDs) vs biologics vs no-csDMARDs treatment) were determined. Synovial tissue was retrieved by minimally invasive, ultrasound-guided biopsy and underwent processing for immunohistochemical (IHC) and molecular characterisation. Samples were analysed for macrophage, plasma-cell and B-cells and T-cells markers, pathotype classification (lympho-myeloid, diffuse-myeloid or pauci-immune) by IHC and gene expression profiling by Nanostring. RESULTS: 128/200 patients were classified as RA1987, 25 as RA2010 and 47 as UA. Patients classified as RA1987 criteria had significantly higher levels of disease activity, histological synovitis, degree of immune cell infiltration and differential upregulation of genes involved in B and T cell activation/function compared with RA2010 or UA, which shared similar clinical and pathobiological features. At 12-month follow-up, a significantly higher proportion of patients classified as lympho-myeloid pathotype required biological therapy. Performance of a clinical prediction model for biological therapy requirement was improved by the integration of synovial pathobiological markers from 78.8% to 89%-90%. CONCLUSION: The capacity to refine early clinical classification criteria through synovial pathobiological markers offers the potential to predict disease outcome and stratify therapeutic intervention to patients most in need.

Synovial cellular and molecular signatures stratify clinical response to csDMARD therapy and predict radiographic progression in early rheumatoid arthritis patients.

OBJECTIVES: To unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally. METHODS: 144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression. RESULTS: Cellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) diffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression. CONCLUSIONS: We demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes.

Validity of a two-component imaging-derived disease activity score for improved assessment of synovitis in early rheumatoid arthritis.

OBJECTIVES: Imaging of joint inflammation provides a standard against which to derive an updated DAS for RA. Our objectives were to develop and validate a DAS based on reweighting the DAS28 components to maximize association with US-assessed synovitis. METHODS: Early RA patients from two observational cohorts (n = 434 and n = 117) and a clinical trial (n = 59) were assessed at intervals up to 104 weeks from baseline; all US scans were within 1 week of clinical exam. There were 899, 163 and 183 visits in each cohort. Associations of combined US grey scale and power Doppler scores (GSPD) with 28 tender joint count and 28 swollen joint count (SJC28), CRP, ESR and general health visual analogue scale were examined in linear mixed model regressions. Cross-validation evaluated model predictive ability. Coefficients learned from training data defined a re-weighted DAS28 that was validated against radiographic progression in independent data (3037 observations; 717 patients). RESULTS: Of the conventional DAS28 components only SJC28 and CRP were associated with GSPD in all three development cohorts. A two-component model including SJC28 and CRP outperformed a four-component model (R2 = 0.235, 0.392, 0.380 vs 0.232, 0.380, 0.375, respectively). The re-weighted two-component DAS28CRP outperformed conventional DAS28 definitions in predicting GSPD (Δtest log-likelihood <-2.6, P < 0.01), Larsen score and presence of erosions. CONCLUSION: A score based on SJC28 and CRP alone demonstrated stronger associations with synovitis and radiographic progression than the original DAS28 and should be considered in research on pathophysiological manifestations of early RA. Implications for clinical management of RA remain to be established.

Development of semiquantitative ultrasound scoring system to assess cartilage in rheumatoid arthritis.

OBJECTIVES: To develop and test the reliability of a new semiquantitative scoring system for the assessment of cartilage changes by ultrasound in a web-based exercise as well as a patient exercise of patients with RA. METHODS: A taskforce of the Outcome Measures in Rheumatology Ultrasound Working Group performed a systematic literature review on the US assessment of cartilage in RA, followed by a Delphi survey on cartilage changes and a new semiquantitative US scoring system, and finally a web-based exercise as well as a patient exercise. For the web-based exercise, taskforce members scored a dataset of anonymized static images of MCP joints in RA patients and healthy controls, which also contained duplicate images. Subsequently, 12 taskforce members used the same US to score cartilage in MCP and proximal interphalangeal joints of six patients with RA in in a patient reliability exercise. Percentage agreement and prevalence of lesions were calculated, as intrareader reliability was assessed by weighted kappa and interreader reliability by Light's kappa. RESULTS: The three-grade semiquantitative scoring system demonstrated excellent intrareader reliability (kappa: 0.87 and 0.83) in the web-based exercise and the patient exercise, respectively. Interreader reliability was good in the web-based exercise (kappa: 0.64) and moderate (kappa: 0.48) in the patient exercise. CONCLUSION: Our study demonstrates that ultrasound is a reliable tool for evaluating cartilage changes in the MCP joints of patients with RA and supports further development of a new reliable semiquantitative ultrasound scoring system for evaluating cartilage involvement in RA.

Characterisation of a solvent-tolerant haloarchaeal (R)-selective transaminase isolated from a Triassic period salt mine.

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.

Characterization of a novel ω-transaminase from a Triassic salt mine metagenome.

Chiral amines are valuable building blocks for the pharmaceutical industry, and are increasingly synthesized by transaminase-mediated (TAm) synthesis. Currently available TAms, primarily isolated from the genomes of cultured mesophilic bacteria, often suffer from a number of drawbacks, including poor substrate range and an inability to tolerate the harsh conditions often demanded by industrial processes. These characteristics have, in part, driven the search for novel biocatalysts from both metagenomic sources and extreme environments. Herein, we report the isolation and characterization of an ω-TAm from a metagenome of a Triassic salt mine in Kilroot, N. Ireland, an extremely hypersaline environment formed circa 220-250 mya. The gene sequence was identified based on homology with existing bacterial TAms, synthesized within a pET28a(+) plasmid and expressed in E. coli BL21 DE3 cells. The resultant 49 kDa protein accepted (S)-methylbenzylamine (MBA) as amino donor and had a specific activity of 0.54 U/mg using α-ketoglutarate (ΑKG) as substrate. Molecular modeling and substrate docking indicated the presence of key residues, conserved in a number of other TAms. Despite the hypersaline environment from which it was isolated, the enzyme displayed low halotolerance, highlighting that not all biocatalysts will demonstrate the extreme characteristics associated with their source environment. This study does however reinforce the viability of mining metagenomic datasets as a means of discovering novel and functional biocatalysts, and adds to a currently scant list of such examples in the field of TAms.

Salinity pulses interact with seasonal dry-down to increase ecosystem carbon loss in marshes of the Florida Everglades.

Coastal wetlands are globally important sinks of organic carbon (C). However, to what extent wetland C cycling will be affected by accelerated sea-level rise (SLR) and saltwater intrusion is unknown, especially in coastal peat marshes where water flow is highly managed. Our objective was to determine how the ecosystem C balance in coastal peat marshes is influenced by elevated salinity. For two years, we made monthly in situ manipulations of elevated salinity in freshwater (FW) and brackish water (BW) sites within Everglades National Park, Florida, USA. Salinity pulses interacted with marsh-specific variability in seasonal hydroperiods whereby effects of elevated pulsed salinity on gross ecosystem productivity (GEP), ecosystem respiration (ER), and net ecosystem productivity (NEP) were dependent on marsh inundation level. We found little effect of elevated salinity on C cycling when both marsh sites were inundated, but when water levels receded below the soil surface, the BW marsh shifted from a C sink to a C source. During these exposed periods, we observed an approximately threefold increase in CO2 efflux from the marsh as a result of elevated salinity. Initially, elevated salinity pulses did not affect Cladium jamaicense biomass, but aboveground biomass began to be significantly decreased in the saltwater amended plots after two years of exposure at the BW site. We found a 65% (FW) and 72% (BW) reduction in live root biomass in the soil after two years of exposure to elevated salinity pulses. Regardless of salinity treatment, the FW site was C neutral while the BW site was a strong C source (-334 to -454 g C·m-2 ·yr-1 ), particularly during dry-down events. A loss of live roots coupled with annual net CO2 losses as marshes transition from FW to BW likely contributes to the collapse of peat soils observed in the coastal Everglades. As SLR increases the rate of saltwater intrusion into coastal wetlands globally, understanding how water management influences C gains and losses from these systems is crucial. Under current Everglades' water management, drought lengthens marsh dry-down periods, which, coupled with saltwater intrusion, accelerates CO2 loss from the marsh.