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Kohei Kubota,1,2 Kohei Onishi,3 Kazuaki Sawaki,3 Tianshu Li,4 Kaoru Mitsuoka,5 Takaaki Sato,6 Shinji Takeoka1,3,4 1Cooperative Major in Advanced Biomedical Sciences, Graduate School of Advanced Sciences and Engineering, Waseda University (TWIns), Tokyo, Japan; 2Formulation Research and Phramaceutical Process Group, CMC R&D Center, Kyowa Hakko Kirin Co., Ltd, Shizuoka, Japan; 3Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering,Waseda University (TWIns), Tokyo, Japan; 4Research Organization for Nano and Life Innovation, Waseda University (TWIns), Tokyo, Japan; 5Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan; 6Department of Chemistry and Materials, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan Abstract: Two lipid-based nanoformulations have been used to date in clinical studies: lipoplexes and lipid nanoparticles (LNPs). In this study, we prepared small interfering RNA (siRNA)-loaded carriers using lipid components of the same composition to form molecular assemblies of differing structures, and evaluated the impact of structure on cellular uptake and immune stimulation. Lipoplexes are electrostatic complexes formed by mixing preformed cationic lipid liposomes with anionic siRNA in an aqueous environment, whereas LNPs are nanoparticles embedding siRNA prepared by mixing an alcoholic lipid solution with an aqueous siRNA solution in one step. Although the physicochemical properties of lipoplexes and LNPs were similar except for small increases in apparent size of lipoplexes and zeta potential of LNPs, siRNA uptake efficiency of LNPs was significantly higher than that of lipoplexes. Furthermore, in the case of LNPs, both siRNA and lipid were effectively incorporated into cells in a co-assembled state; however, in the case of lipoplexes, the amount of siRNA internalized into cells was small in comparison with lipid. siRNAs in lipoplexes were thought to be more likely to localize on the particle surface and thereby undergo dissociation into the medium. Inflammatory cytokine responses also appeared to differ between lipoplexes and LNPs. For tumor necrosis factor-α, release was mainly caused by siRNA. On the other hand, the release of interleukin-1β was mainly due to the cationic nature of particles. LNPs released lower amounts of tumor necrosis factor-α and interleukin-1β than lipoplexes and were thus considered to be better tolerated with respect to cytokine release. In conclusion, siRNA-loaded nanoformulations effect their cellular uptake and immune stimulation in a manner that depends on the structure of the molecular assembly; therefore, nanoformulations should be optimized before extending studies into the in vivo environment. Keywords: nanoformulation, siRNA, cryo-TEM, confocal microscopy, endocytosis, immune stimulation

Athletes become increasingly susceptible to infection with intense exercise, which has an immunosuppressive effect. Sixteen long-distance runners participated in the present study. The clinical indexes, mainly related to the immune state, of 8 subjects in the amino acid intake group and 8 subjects in the placebo intake group were measured before and after a 7-day training camp. Significant increases in levels of myoglobin (Mb) and growth hormone (GH), and significant decreases in natural killer cell activity (NK) and Fisher's ratio were observed after as compared to before the training camp in the placebo group. However, these changes were not observed in the amino acid supplementation group. These findings suggest that amino acid supplementation during a training camp may have a stress-reducing effect by inhibition of muscle protein degradation, as well as by inhibiting the reduction of immunological functions, and may also contribute to protection against infectious diseases. [ABSTRACT FROM AUTHOR]

< MRI for stress fracture > Early diagnosis and treatment for stress fractures are important to enable a return to full athletic activities as soon as possible. We investigated the early diagnostic efficacy of MRI for stress fractures. Subjects were 38 patients (39 limbs)whose ages ranged from 12 to 27 years(mean, 20 years). Each patient was examined by MRI, plain radiography, and bone scintigraphy during the same period. The signal change was more evident in the femur and lateral malleolus and tarsal navicular bone, but, signal changes of tibia stress fractures were varied. The subjects examined by MRI within 2 weeks from onset showed obvious signal change, but their plain radiograms at the same time revealed no abnormalities. These subjects diagnosed in the early stage by MRI could return to sports earlier than those diagnosed 4 or more weeks after onset. < Bone metabolism markers(BMM)and bone mineral density(BMD)for stress fracture > We investigated the bone metabolism markers(BMM)and bone mineral density(BMD)in female long-distance runners of a university. Subjects were 14 female runners. As BMM, we investigated PICP, osteocalcin, AL-P(bone), DPD, and NTx. As BMD, femoral neck, knee and lumber vertebrae were investigated at the stage between rest and hard training. In runners with amenorrhea or with stress fracture, NTx was markedly higher and BMD was lower than in female runners with normal menstruation. These factors are good markers for studying the condition of female runners. [ABSTRACT FROM AUTHOR]

1. This study examined the effect of diazepam (DZP) on phosphoinositide turnover, which plays an important role in the regulation of salivary secretion, in rat parotid acinar cells. 2. DZP (10(-9) M to 10(-5) M), a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose-dependently decreased inositol 1,4,5-trisphosphate IP3 production stimulated by carbachol, a muscarinic receptor agonist, in the cells. 3. DZP produced a maximum inhibitory response at a concentration of 10(-5) M, with IP3 production decreased to 63% of maximal levels. The concentration inducing half maximal inhibition of IP3 production was approximately 3.5 x 10 (-8) M. 4. An inhibitory response to DZP was produced by a short-term pretreatment (<3 min) of the cells and prevented by antagonist and competing ligand for the central- and peripheral-type benzodiazepine receptors, flumazenil and PK 11195, respectively. 5. DZP showed a non-competitive inhibition of carbachol-stimulated IP3 production. It did not directly inhibit the activities of GTP-binding regulatory proteins and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) in the parotid gland membranes, though choline chloride inhibited PLC activity. 6. DZP (10(-5) M) attenuated the increase in the intracellular Ca2+ concentration ([Ca(2+)](i)) in the cells following stimulation of the muscarinic and alpha(1)-adrenoceptors. 7. These results suggest that in the parotid acinar cells, DZP inhibits muscarinic receptor-stimulated IP3 production through benzodiazepine receptors and that PLC activity which produces IP3 is inhibited by chloride. The decreases in IP3 and [Ca(2+)](i) in the cells may be connected with the suppression of salivary secretion induced by DZP. [ABSTRACT FROM AUTHOR]