Specific residues at every third position of siRNA shape its efficient RNAi activity

Small interfering RNA (siRNA) induces sequence-specific post-transcriptional gene silencing in mammalian cells. Different efficacy of each siRNA is considered to result from sequence preference by protein components in RNAi. To obtain mechanistic insight into siRNA functionality, here we describe a complete data set of siRNA activities targeting all possible position of a single mRNA in human cells. Seven hundred and two siRNAs covering open reading frame of enhanced green fluorescent protein mRNA ( 720 bases) were examined with minimized error factors. The most important finding is that specific residues at every third position of siRNAs greatly influence its RNAi activity; the optimized base composition at positions 3n + 1 (4,7,10,13,16,19) in siRNAs have positive effects on the activity, which can explain the waving siRNA activity with 3 nucleotides (nt) periodicity in the sequential positions of mRNAs. Since there was an obvious correlation between siRNA activity and its binding affinity to TRBP, a partner protein of human Dicer, the 3-nt periodicity might correlate with the affinity to TRBP. As an algorithm (‘siExplorer’) developed by this observation successfully calculated the activities of siRNAs targeting endogenous human genes, the 3-nt periodicity provides a new aspect unveiling siRNA functionality

Liquid-Liquid Interface Can Promote Micro-Scale Thermal Marangoni Convection in Liquid Binary Mixtures

Liquid-liquid phase separation, a physical transition in which a homogeneous solution spontaneously demixes into two coexisting liquid phases, has been a key topic in the thermodynamics of two-component systems and may find applications in separation, drug delivery, and protein crystallization. Here we applied a microscale temperature gradient using optothermal heating of a gold nanoparticle to overcome the experimental difficulties inherent in homogeneous heating: we aimed at highlighting precise structural development by avoiding randomly nucleating/growing microdomains. In response to laser illumination, a single gold nanoparticle immersed in a binary mixture of aqueous 2,6-dimethylpiridine (lutidine) and N-isopropylpropionamide (NiPPA) was clearly sensitive to the phase transition of the surrounding liquid as demonstrated by light scattering signals, spectral red-shifts and bright-spot images. The local phase separation encapsulating the gold nanoparticle resulted in immediate formation and growth of an organic-rich droplet which was confirmed by Raman spectroscopy. Remarkably, the droplet was stable under a non-equilibrium steady-state heating condition because of strong thermal confinement. Microdroplet growth was ascribed to thermocapillary flow induced by a newly formed liquid-liquid interface around the hot gold nanoparticle. Based upon a tracer experiment and numerical simulation, it is deduced that the transport of solute to the high temperature area is driven by this thermocapillary flow. This study enhances our understanding of phase separation in binary mixtures induced by microscale temperature confinement

Obstructions of Portal Veins and Tumor Numbers Are Associated with Humped Hepatocellular Carcinoma

Tumor protrusion in hepatocellular carcinoma (HCC) is one of the risk signs of tumor rupture. Despite curative tumor treatments, HCC recurrences sometimes occur with rapidly growing humped or ruptured HCC in small sized tumors. The aim of this study was to clarify the characteristics of humped HCC clinically and radiologically associated with tumor progression, liver damage, and treatment. The subjects were 179 consecutive HCC patients who underwent angiographic examination. Dynamic studies, using helical computed tomography and magnetic resonance imaging were assessed, and the HCC area were measured. The tumor-node-metastasis (TNM) stage differed significantly between the humped and non-humped HCC groups. Humped HCC was more frequently observed in the right lobe (29.3% of right-lobe HCCs) than in the left (10.1%;p＝0.003). Analysis of recurrent HCC revealed that patients with multiple treatments of >4 sessions had more humped HCC (33.8%) than those with 1-3 sessions (16.7%;p＝0.042). Multivariate regression analysis revealed that tumor invasion in the portal vein, rather than large tumor size, was significantly associated with tumor protrusion. HCC recurrence with humped HCC occurs often in patients with multiple treatments. Tumor factors of the TNM classification, especially tumor invasion in the portal vein, might be associated with the mechanisms of tumor protrusion

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Delta orf1 and Delta orf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Delta orf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Delta orf1, Delta orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule

Association of lower limb muscle mass and energy expenditure with visceral fat mass in healthy men

BACKGROUND: A high-calorie diet and physical inactivity, an imbalance between caloric intake and energy consumption, are major causes of metabolic syndrome (MetS), which manifests as accumulation of visceral fat and insulin resistance. However, the lifestyle-related factors associated with visceral fat mass in healthy men are not fully understood. METHODS: We evaluated visceral fat area (VFA), skeletal muscle mass, caloric intake, and energy expenditure in 67 healthy male participants (mean age, 36.9 ± 8.8 years; body mass index 23.4 ± 2.5 kg/m(2)). RESULTS: Multiple regression analysis showed that the total skeletal muscle mass (P < 0.001) were negatively and age (P < 0.001) were positively associated with VFA. Lower limb muscle mass (P < 0.001) was strongly associated with VFA. However, total caloric intake, total energy expenditure, and energy expenditure during exercise were not associated with VFA. CONCLUSIONS: Skeletal muscle mass especially lower limb muscle mass negatively contributes to visceral fat mass in healthy men. Therefore, maintaining lower limb muscular fitness through daily activity may be a useful strategy for controlling visceral obesity and metabolic syndrome