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BackgroundD-Dimer testing is a diagnostic tool for exclusion of deep vein thrombosis (DVT) and pulmonary embolism (PE). This study evaluated the diagnostic performance of the Tina-quant® D-Dimer Gen.2 assay (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) in patients with low/intermediate pre-test probability of DVT/PE using standard, age-, and clinical probability-adjusted cut-offs.MethodsIn this prospective, observational, multicenter study (July 2017–August 2019), plasma samples were collected from hospital emergency departments and specialist referral centers. DVT/PE was diagnosed under hospital standard procedures and imaging protocols. A standard D-dimer cut-off of 0.5 µg fibrinogen equivalent units (FEU)/ml was combined with the three-level Wells score; cut-offs adjusted for age (age × 0.01 µg FEU/ml for patients >50 years) and clinical probability (1 µg FEU/ml for low probability) were also evaluated. An assay comparison was conducted in a subset of samples using the Tina-quant D-Dimer Gen.2 assay and the previously established routine laboratory assay, STA-Liatest D-Di Plus assay (Stago Deutschland GmbH, Düsseldorf, Germany).Results2,897 patients were enrolled; 2,516 completed the study (DVT cohort: 1,741 PE cohort: 775). Clinical assessment plus D-dimer testing using the standard cut-off resulted in 317 (DVT) and 230 (PE) false positives, and zero (DVT) and one (PE) false negatives. Negative predictive value (NPV) was 100.0% (95% confidence interval [CI]: 99.7%–100.0%) and 99.8% (95% CI: 98.8%–100.0%) for DVT and PE, respectively. After age-adjustment, NPV was 99.9% (95% CI: 99.6%–100.0%) and 99.1% (95% CI: 97.8–99.7) for DVT and PE, respectively. False positive rates decreased (>50%) in clinical probability-adjusted analyses vs. primary analysis. In the assay comparison, the performances of the two assays were comparable.ConclusionThe Tina-quant D-Dimer Gen.2 assay and standard D-dimer cut-off level combined with the three-level Wells score accurately identified patients with a very low probability of DVT/PE.

Toxin A (TcdA) and B (TcdB) from Clostridium difficile enter host cells by receptor-mediated endocytosis. A prerequisite for proper toxin action is the intracellular release of the glucosyltransferase domain by an inherent cysteine protease, which is allosterically activated by inositol hexaphosphate (IP6). We found that in in vitro assays, the C-terminally-truncated TcdA1–1065 was more efficient at IP6-induced cleavage compared with full-length TcdA. We hypothesized that the C-terminally-located combined repetitive oligopeptides (CROPs) interact with the N-terminal part of the toxin, thereby preventing autoproteolysis. Glutathione-S-transferase (GST) pull-down assays and microscale thermophoresis confirmed binding between the CROPs and the glucosyltransferase (TcdA1–542) or intermediate (TcdA1102–1847) domain of TcdA, respectively. This interaction between the N- and C-terminus was not found for TcdB. Functional assays revealed that TcdB was more susceptible to inactivation by extracellular IP6-induced cleavage. In vitro autoprocessing and inactivation of TcdA, however, significantly increased, either by acidification of the surrounding milieu or following exchange of its CROP domain by the homologous CROP domain of TcdB. Thus, TcdA CROPs contribute to the stabilization and protection of toxin conformation in addition to function as the main receptor binding domain.

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.