Anticoagulants and the propagation phase of thrombin generation

The view that clot time-based assays do not provide a sufficient assessment of an individual''s hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants.     

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.     

A new tool for biotechnology: AdoMet-dependent methyltransferases

AdoMet-dependent methyltransferases catalyze highly specific methyl group transfers from the ubiquitous cofactor S-adenosyl-L-methionine to a multitude of biological targets in the cell. Recently, DNA methyltransferases have been used for the sequence-specific, covalent attachment of larger chemical groups to plasmid and bacteriophage DNA using two classes of synthetic AdoMet analogs. These synthetic cofactors, in combination with the myriad AdoMet-dependent methyltransferases available in nature, provide new molecular tools for precise, targeted functionalization and labeling of large natural DNAs and, in all likelihood, RNAs and proteins. This paves the way for numerous novel applications in the functional analysis of biological methylation, biotechnology and medical diagnostics.     

Recognition of guanosine by dissimilar tRNA methyltransferases

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.     

Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases

Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2'-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.     

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.     

A comparison of two different anthelmintic treatment regimens against natural gastrointestinal nematode infections on two Lithuanian sheep farms

Background

According to targeted treatment (TT), the whole flock is dewormed based on knowledge of the risk, or parameters that quantify the mean level of infection, whereas according to targeted selective treatment (TST), only individual animals within the grazing group are treated, based on parasitological, production and/or morbidity parameters. The aim of this study was to compare two different treatment protocols on sheep farms in Lithuania. The study was conducted from 15 April to 31 October 2014 on three sheep farms. On the TT (the whole flock) and T(S)T (with FECs ≥ 300, respectively) farms all adult animals were treated orally with fenbendazole irrespective of EPG counts before the grazing season. The second treatment was applied with injectable ivermectin on both farms. However, on the TT farm all sheep were also treated on 2nd August regardless of their EPG counts, while on the T(S)T farm only those animals with an EPG ≥ 300 were treated on 1 July using a threshold of ≥ 300 EPG. No treatments were administered on the control farm (n = 1) during the study.

Results

Spring treatment of ewes significantly reduced nematode faecal egg counts (FEC) both on the TT and T(S)T farms, with the benefit of lowering pasture contamination with infective L₃ stage larvae at the start of grazing season, while it remained significantly higher on the control farm. The positive effect of the spring treatment of ewes was reflected by increased body weight gains (BWG) in lambs in the first half of the grazing season. Following the second treatment, the weight gains in lambs on the T(S)T farm were higher compared to lambs on the TT farm, while BWG in the control lambs started to decrease. The difference was also substantiated by the body condition scores (BCS) and dag scores (DS) of lambs, which were highest on the T(S)T farm compared with those on the control and TT farms.

Conclusions

The results of this study show that both treatment strategies were useful in reducing clinical effects (BCS and DS) of gastrointestinal nematode parasitism and increasing the performance in lambs. Furthermore, on the T(S)T farm some of animals were left in refugia, helping to slow down the development of anthelmintic resistance (AR) in future.