Inferred amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from five different methanogen species were aligned and two regions with a high degree of homology flanking a more variable region were identified. Analysis of the DNA sequences from the conserved regions yielded two degenerate sequences from which a forward primer, a 32-mer, and a reverse primer, a 23-mer, could be derived for use in the specific PCR-based detection of methanogens. The primers were successfully evaluated against 23 species of methanogen representing all five recognized orders of this group of Archaea, generating a PCR product between 464 and 491 bp. Comparisons between the mcrA and 16S small subunit rRNA gene sequences using PHYLIP demonstrated that the tree topologies were strikingly similar. Methods were developed to enable the analysis of methanogen populations in landfill using the mcrA gene as the target. Two landfill sites were examined and 63 clones from a site in Mucking, Essex, and 102 from a site in Odcombe, Somerset, were analysed. Analysis revealed a far greater diversity in the methanogen population within landfill material than has been seen previously.

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One strain of a thermophilic, slightly halotolerant bacterium was isolated from a thermally polluted industrial runoff near Salisbury, United Kingdom. This organism, strain PRD-lT (T = type strain), for which we propose the name Rubrobacter xylanophilus sp. nov., produces short gram-positive rods and coccoid cells and forms pink colonies. The optimum growth temperature is approximately 60°C. Unusual internal branchedchain fatty acids (namely, 12-methylhexadecanoic acid and 14-methyloctadecanoic acid) make up the major acyl chains of the lipids. The results of our 16s rRNA sequence comparisons showed that strain PRD-lT is related to Rubrobacter radiotolerans and that these two organisms form a deep evolutionary line of descent within the gram-positive Bacteria.Over the past 20 years the microbiology of thermophiles has been dominated by the isolation and characterization of thermophilic Archaea species, many of which grow at extreme temperatures. During this time, however, many new thermophilic Bacteria species, several belonging to the gram-positive phylum, have been isolated from a wide range of thermal environments. The majority of the thermophilic gram-positive Bacteria species that have been described belong to the genera Bacillus, Alicyclobacillus, and Clostridium and other recently proposed genera, some of which were formerly included in the genus Clostridium (3,5,18,28,33,40).The species Rubrobacter radiotolerans, which was initially named Arthrobacter radiotolerans, was described on the basis of one strain isolated from a hot spring in Japan after gamma irradiation of water samples (35,44). R. radiotolerans is gram positive, has an optimum growth temperature of about 48"C, and forms short pleomorphic rod-shaped cells. It is also highly radiotolerant and possesses unique internally branched fatty acids.A pink-pigmented strain was recently isolated from a thermally polluted runoff from a carpet factory in the United Kingdom. This organism is more thermophilic than R. radiotolerans and also possesses unusual branched-chain fatty acids. On the basis of morphological and biochemical characteristics, chemotaxonomic parameters, and 16s rRNA gene sequence data, we propose that this organism, strain PRD-lT (T = type strain), belongs to a new species, Rubrobacter xylanophilus. MATERIALS AND METHODSIsolation of strain PRD-IT. Strain PRD-lT was isolated from thermally polluted runoff (temperature, 50°C) from a carpet factory in Wilton, Wiltshire, United Kingdom. This strain was isolated by spreading a biofilm sample with a glass rod on tryptone soya agar. After incubation at 50°C for 5 days, one pinkpigmented colony appeared on a culture plate and was purified on the same medium. R. radiotolerans DSM 46359T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. Strains PRD-lT and DSM 46359T were routinely grown in Thermus medium containing 1.0 g of yeast extract per liter and 1.0 g of tryptone medium (41) per liter and were stored at -80°C in the same medium containing 1...

Simulation exercises are an important part of emergency preparedness activities for the healthcare community but evidence of their impact on the response to real major incidents is limited. This project studied the impact of health emergency preparedness exercises (HEPEs) on the response to a mass casualty terrorist incident. The mixed methods study design was adopted comprising an on-line survey and follow up individual interviews. Participants were healthcare staff who took part in responses to three major terrorist incidents in the UK in 2017. Descriptive statistics and analysis of variance were undertaken with quantitative data. Content and thematic analysis methods were used for qualitative data analysis. The online survey generated 86 responses; 79 (92%) were from the responders to the Manchester Arena bombing. Twenty-one survey respondents shared their experiences in in-depth interviews. Healthcare staff who took part in HEPEs felt better prepared to respond than those who did not attend an exercise. The most commonly reported benefits from HEPEs were awareness of major incident plans and having the opportunity to practice responding to a similar scenario in the recent exercise. Specific benefits included: improved coordination of the response through adherence to recently practiced incident plans ; confidence with response roles; real-time modifications of the response and support provided to staff who did not take part in exercises. Exercise recency was highlighted as an important facilitating factor. The study provides strong objective evidence that the response to a mass casualty terrorist incident was enhanced by training and service development achieved through HEPEs.

IntroductionSystem learning from major incidents is a crucial element of improving preparedness for response to any future incidents. Sharing good practice and limitations stimulates further actions to improve preparedness and prevents duplicating mistakes.MethodsThis convergent parallel mixed methods study comprises data from responses to an online survey and individual interviews with healthcare staff who took part in the responses to three terrorist incidents in the UK in 2017 (Westminster Bridge attack, Manchester Arena Bombing and London Bridge attack) to understand limitations in the response and share good practices.ResultsThe dedication of NHS staff, staff availability and effective team work were the most frequently mentioned enabling factors in the response. Effective coordination between teams and a functional major incident plan facilitated an effective response. Rapid access to blood products, by positioning the blood bank in the ED, treating children and parents together and sharing resources between trauma centres were recognised as very effective innovative practices. Recent health emergency preparedness exercises (HEPEs) were valued for preparing both Trusts and individual staff for the response. Challenges included communication between ambulance services and hospitals, difficulties with patient identification and tracking and managing the return to ‘normal’ work patterns post event. Lack of immediately available clinical protocols to deal with blast injuries was the most commonly mentioned clinical issue. The need for psychosocial support for responding and supporting staff was identified.DiscussionBetween-agencies communication and information sharing appear as the most common recurring problems in mass casualty incidents (MCIs). Recent HEPEs, which allowed teams, interdisciplinary groups, and different agencies to practice responding to similar simulated incidents, were important and informed actions during the real response. Immediate and delayed psychosocial support should be in place for healthcare staff responding to MCIs.

Introduction Fibrin monomer (FM) concentrations reflect pro-thrombin activity and have the potential to predict thrombotic events relatively earlier than other haemostatic markers. Most often, FM are compared with D-dimer (DD) as increased DD have been documented in disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT) and pulmonary embolism. Although DD have a high sensitivity and negative predictive value, their specificity is much lower depending on the assay chosen, clinical pre-test probability and patient condition. There are limited reports investigating the utility of FM in hyper-coagulable patients. Methods We performed a literature search of FM concentrations in hyper-coagulable patients including those with DIC, acute ischaemic stroke, atrial fibrillation, acute myocardial infarction, venous thromboembolism (VTE) and cancer, as well as those who are pregnant or undergoing surgery. Results FM were increased in patients with DIC and those with malignancy. In contrast, detection of VTE or post-operative DVT development is likely enhanced using both FM and DD concentrations. Similarly, measuring FM concentrations with other biomarker levels may be more beneficial in patients suffering an acute myocardial infarction or acute ischaemic stroke. Lastly, FM concentrations vary substantially throughout pregnancy with no definitive role of FM as of yet. Conclusion Utilizing FM concentrations to assess hyper-coagulable patients seems promising; however, there are limitations including variations in FM cut-off values, the effect of patient medications and the timing of FM measurement relative to an acute event. Thus, further investigation is required before a true advantage for FM as a haemostatic marker can be established.

Essentials The value of thrombin generation assay (TGA) in monitoring direct oral anticoagulants (DOAC) is not well defined.TGA parameters were measured and correlated to DOAC levels in 10 healthy volunteers after oral intake of DOACs.Lag time is the only sensitive TGA parameter across different DOACs, dabigatran, rivaroxaban and apixaban.Endogenous thrombin potential had weak correlation with DOAC levels and not suitable as stand‐alone parameter. BackgroundThere are clinical situations where monitoring direct oral anticoagulants (DOACs) may be useful. The clinical application of thrombin generation assay (TGA) in monitoring the effect of DOACs has not been well established. An ex vivo study was performed to systematically evaluate the anticoagulant effect of dabigatran, rivaroxaban and apixaban on each individual TGA parameter through serial measurements over time to assess suitability of these parameters for monitoring the anticoagulant effect of DOACs.MethodsTen healthy volunteers were given oral dabigatran 150 mg, rivaroxaban 20 mg, or apixaban 10 mg once. TGA parameters lag time, endogenous Thrombin potential (ETP), and thrombin peak height, time to peak, and velocity index were measured at times 0, 2, 4, and 24 hours after intake of DOAC. TGA parameters and DOAC concentrations were correlated.ResultsThe lag time was significantly correlated with all DOAC concentrations (r ≥ .81, P < .0001 for all). Thrombin peak height best correlated with direct Factor Xa inhibitor (FXa) concentrations in nonlinear fashion (R² ≥ .87). ETP was weakly correlated with DOAC levels (r ≤ .68). Besides lag time, the other TGA parameters were not significantly altered over time by dabigatran.ConclusionLag time was the only sensitive TGA parameter across the different classes of DOACs evaluated. Thrombin peak height was strongly correlated to FXa inhibitor concentrations and potentially a useful parameter to monitor FXa inhibitors at low concentrations. ETP had a weak correlation with achieved DOAC concentrations and is likely less suitable for assessment of DOAC effect as a stand‐alone parameter.

Factor XI (FXI) is a homodimeric blood coagulation protein. Each monomer comprises four tandem apple-domain repeats (A1-A4) and a serine protease domain. We report here the NMR solution structure of the A4 domain (residues 272-361), which mediates formation of the disulfide-linked FXI dimer. A4 exhibits characteristic features of the plasminogen apple nematode domain family, including a five-stranded ␤-sheet flanked by an ␣-helix on one side and a two-stranded ␤-sheet on the other. In addition, the solution structure reveals a second ␣-helix at the C terminus. Comparison with a recent crystal structure of full-length FXI, combined with molecular modeling, suggests that the C-terminal helix is formed only upon proteolytic activation. The newly formed helix disrupts interdomain contacts and reorients the catalytic domains, bringing the active sites into close proximity. This hypothesis is supported by small-angle x-ray scattering and electron microscopy data, which indicate that FXI activation is accompanied by a major change in shape. The results are consistent with biochemical evidence that activated FXI cleaves its substrate at two positions without release of an intermediate.blood coagulation ͉ NMR ͉ plasminogen apple nematode domain ͉ small-angle x-ray scattering ͉ EM F actor XI (FXI) is a blood plasma protein in the intrinsic pathway of blood coagulation. In response to blood vessel injury, thrombin can convert the homodimeric FXI zymogen into its proteolytically active form, FXIa, which in turn cleaves its substrate, factor IX (FIX), resulting in a cascade of events leading to fibrin formation (1, 2). FXI may also play a role in protection of clots from fibrinolysis (3). The enzymatic activators of FXI (thrombin, FXIIa, or FXIa) cleave the Arg-369-Ile-370 bond in each monomer of FXI, yielding FXIa, which consists of a 369-residue heavy chain and a disulfide-linked serine protease domain of 238 residues. A substantial body of evidence indicates that binding interactions outside the protease domain (exosites) are important determinants of ligand and receptor interactions and of substrate affinity and specificity in coagulation reactions involving FXI/XIa (2).The FXI heavy chain comprises four apple-domain repeats, A1-A4, each of which contains six cysteine residues involved in intradomain disulfide bonds (1, 4). An additional cysteine in A4 (Cys-321) forms a physiologically important disulfide bridge with the corresponding Cys in the other subunit of the FXI dimer. The sequence identity among the four FXI apple domains ranges from 23% to 34%. They are members of the plasminogen apple nematode (PAN) domain family of proteins (5). Various domains of FXI have been modeled by using PAN domain structures as templates (6). However, the model could not predict the quaternary structure of the A4 domain, which mediates dimerization of FXI through both noncovalent interactions as well as an intersubunit disulfide bond (Cys-321-Cys-321Ј). A recent crystal structure of the FXI zymogen (7) showed that all of the contacts ...