Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

A Case of Closed Pilon Fracture Resulting in Soft-Tissue Necrosis and Treated with Reverse Sural Artery Flap and Circular External Fixation.

Tibial pilon fractures are difficult to treat due to articular comminution and soft-tissue injuries caused by high-energy trauma. Open reduction and internal fixation is a commonly used method of treatment. However, it has a high risk of infection and soft-tissue complications due to the extensive detachment of soft tissue. We report on a case with a tibial pilon fracture and soft-tissue necrosis that we treated using limited internal fixation combined with a circular external fixator (LIFCEF) and reverse sural artery flap (RSAF) as part of an orthoplastic approach within the orthopedic surgery department alone, which obtained good results. A 51-year-old man was injured in a motorcycle accident and transported to a nearby hospital. X-rays at the time of injury showed tibial pilon fractures (AO Foundation/Orthopedic Trauma Association 43c3.3, Ruedi-Allgower: Type III). Soft-tissue necrosis with blisters on the medial side of the lower leg (AO soft-tissue classification: IC3-MT1-NV1) was observed. In addition, the patient was referred to our hospital on day 10 of the injury. LIFCEF was chosen for treating the fracture because plate fixation was accompanied by the risk of plate exposure, soft-tissue complications, and an increased skin defect area, and RSAF was chosen to reconstruct the soft tissue defect. Four years after the surgery, the American Orthopedic Foot and Ankle Score was 92 points. X-ray alignment evaluation showed mLDTA 93° and aADTA 91°. Stage 2 arthrosis was present according to the Takakura ankle osteoarthritis classification, but the patient was able to walk without pain. Tibial pilon fractures are difficult to treat due to articular comminution and soft-tissue injuries caused by high-energy trauma. The timing and choice of treatment are crucial concerning the soft tissue.

Synthesis of Resolvin E1 and Its Conformationally Restricted Cyclopropane Congeners with Potent Anti-Inflammatory Effect.

RvE1 (1) is an endogenous lipid mediator with very potent anti-inflammatory activity, which is due to the inhibition of neutrophil chemotaxis and inflammatory cytokine production and the promotion of macrophage phagocytosis. On the basis of the conformational analysis of RvE1, we designed its four cyclopropane congeners (2a-d), in which the conformationally flexible terminal C1-C4 moiety of RvE1 was rigidified by introducing stereoisomeric cyclopropanes. The four congeners and also RvE1 were efficiently synthesized via a common synthetic route. The evaluation of the anti-inflammatory effects of the compounds in mice resulted in the identification of trans-ß-CP-RvE1 (2d), which was significantly more active than RvE1, as a potential lead for anti-inflammatory drugs of a novel mechanism of action.

Design and Synthesis of Benzene Congeners of Resolvin E2, a Proresolving Lipid Mediator, as Its Stable Equivalents.

Resolvins (Rvs) are highly potent anti-inflammatory lipid mediators that are chemically and biologically unstable because of their polyunsaturated structures. To address this issue, we designed benzene congeners of RvE2, i.e., o-, m-, and p-BZ-RvE2s, as stable equivalents of RvE2 by replacing the unstable skipped diene moiety with a benzene ring on the basis of computational conformation studies and synthesized these congeners via a short common route through two Stille couplings. o-BZ-RvE2 exhibited more potent anti-inflammatory activity and much higher metabolic stability than RvE2. Thus, o-BZ-RvE2 was identified as a stable equivalent of RvE2, which is useful as a lead for anti-inflammatory drugs with a new mechanism of action as well as a biotool for investigating RvE2-mediated inflammation resolving pathways.