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Abstract: Glucose oxidase (GOx) is an important industrial enzyme and is recognized as the gold standard for monitoring blood glucose. However, due to its inherent oxidase property, the presence of oxygen affects electrochemical measurements of venous blood glucose employing artificial electron mediators. We therefore attempted to engineer Penicillium amagasakiense-derived GOx into a dehydrogenase by focusing on the amino acid residues predicted to interact with oxygen. Our rational amino acid substitution approach resulted in the construction of the Ser114Ala/Phe355Leu mutant, which has an 11-fold decrease in oxidase activity and 2.8-fold increase in dehydrogenase activity compared with wild-type GOx. As a result, the dehydrogenase/oxidase activity ratio of the engineered enzyme was 32-fold greater than that of the wild-type enzyme. The enzyme sensor constructed with Ser114Ala/Phe355Leu was considerably less affected by oxygen than the wild-type GOx-based sensor at lower glucose concentrations. [Copyright &y& Elsevier]

Abstract: Smart organic–inorganic hybrids were prepared using noncovalent interactions between water-soluble silsesquioxane nanoparticles containing tertiary amine moieties and amino acid-based polymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(N-acryloyl-4-trans-hydroxy-l-proline) and poly(N-acryloyl-l-proline), or poly(A-Hyp-OH) and poly(A-Pro-OH), respectively, were employed as weak polyelectrolytes, whereas their methylated versions, poly(A-Hyp-OMe) and poly(A-Pro-OMe), were used as thermoresponsive polymers. Simple mixing of aqueous solutions of poly(A-Hyp-OH) and the silsesquioxane nanoparticles led to a straightforward formation of the pH-responsive hybrids, in which a white turbid dispersion was observed at pH=5–8, whereas transparent solutions were obtained at pH=2 and 10. The aggregate structures and chiroptical properties of the hybrids were evaluated by scanning force microscopy (SFM) and circular dichroism (CD) measurements. Complex formation with poly(A-Pro-OH) and silsesquioxane nanoparticles was also observed in methanol. Thermoresponsive hybrids were obtained by complexation of the silsesquioxane nanoparticles with poly(A-Hyp-OMe) in water, whereas poly(A-Pro-OMe) showed no specific interaction. To the best of our knowledge, this is the first report of the synthesis of smart organic–inorganic hybrids with pH-responsive and thermoresponsive properties that can be manipulated by the complexation of amino acid-based polymers and water-soluble silsesquioxane nanoparticles. [Copyright &y& Elsevier]

Nucleoli are sites where both the large and small ribosomal subunits mature. Biochemical assays have suggested that a multivalent nucleolar protein, NPM1/nucleophosmin contributes to the formation of the outer layer of the nucleolus. Prior works show that NPM1 depletion disorganizes the nucleolar structure. However, the mechanism of how NPM1 regulates the nucleolar structure has been unknown. We demonstrated that NPM1 directly interacts with the large ribosomal subunits and maintains them in the nucleolus. Ectopically localized NPM1 efficiently recruits only the large ribosomal subunit precursors, while ectopically localized large ribosomal subunit by the ribosomal protein RPL4 efficiently recruits NPM1. These results suggest that the nucleolar localization of NPM1 and the large ribosomal subunit precursors are mutually dependent. Furthermore, proteomic and localization analyses suggest that NPM1 plays a crucial role in the accumulation of the late processing machinery of the large ribosomal subunits in the nucleolus. Our results suggest that NPM1 maintains the pre-ribosomes and assembly machinery in the nucleolus, which in turn determines the nucleolar volume. • NPM1 depletion changed the GC region of the nucleolus. • NPM1 directly interacts with the 60S ribosomal subunits. • NPM1 depletion decreased the nucleolar accumulation of the pre-60S subunits and a subset of pre-60S processing factors. • NPM1 depletion inhibits the 32S pre-rRNA processing. [ABSTRACT FROM AUTHOR]

Abstract Background Plasma removal by washing is an effective approach to prevent transfusion reactions by platelet concentrates (PCs). Recently, washed PCs were released by the Japanese Red Cross Society (JRCS). Materials and methods This retrospective multicenter study evaluated the efficacy and safety of released washed PCs (RWPCs) between September 2016 and January 2017 in Japan. The RWPCs were prepared by washing leukoreduced apheresis PCs with the platelet additive solution, BRS-A, using automated cell processors. Results Clinical data were obtained from 91 patients and 1210 RWPC transfusions at 50 institutions. The median number of RWPC transfusions per patient was 8 (range, 1–91). RWPCs were used in 94.5% of the patients with a history of recurrent or severe transfusion reactions for preventing such reactions. Responses of RWPCs were evaluated as complete response (91.6%), partial response (8.2%), no-change (0.2%), and progression (0%) and overall response was equal across subgroups divided by patients' profiles. The median corrected count increment (CCI) at 1 and 24 h post-transfusion were 13.5 (range, 1.9–35.4) × 109/L and 3.5 (range, −13 to 53.6) × 109/L, respectively, and median CCI at 24 h was 5.5 (range, −13 to 53.6) × 109/L in patients without risk factors associated with platelet transfusion refractoriness. Transfusion reactions to RWPCs were observed in only nine transfusions (0.7%), all of which were mild allergic reactions. Conclusion This study demonstrated that RWPCs were effective and safe in patients with a history of transfusion reactions. Further prospective studies on efficacy together with cost-benefit analysis in RWPCs are needed. [ABSTRACT FROM AUTHOR]

Aims T lymphocytes synthesize acetylcholine (ACh) and express muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively) responsible for increases in the intracellular Ca 2 + concentration ([Ca 2 + ] i ). Our aim in the present study was to assess whether autocrine ACh released from T lymphocytes regulates their physiological functions. Main methods MOLT-3 human leukemic cell line and murine splenocytes were loaded with fura-2 to monitor [Ca 2 + ] i changes in the absence or presence of several AChR antagonists, including mecamylamine, methyllycaconitine and scopolamine. Real-time PCR and ELISA were performed to measure interleukin-2 (IL-2) mRNA and protein levels. Key findings T lymphocytes constitutively produce sufficient amounts of ACh to elicit autocrine changes in [Ca 2 + ] i . These autocrine ACh-evoked [Ca 2 + ] i transients were mediated by nAChRs and then influx of extracellular Ca 2 + . Mecamylamine, a nAChR inhibitor, suppressed not only these [Ca 2 + ] i transients, but also IL-2 release and T cell proliferation. Significance Here, we confirmed that T lymphocytes utilize ACh as a tool to interact with each other and that autocrine ACh-activated nAChRs are involved in cytokine release and cell proliferation. These findings suggest the possibility that nAChR agonists and antagonists and smoking are able to modulate immune function, which in turn suggests the therapeutic potential of immune activation or suppression using nAChR agonists or antagonists. [ABSTRACT FROM AUTHOR]