The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices

Eating-related distress and need for nutritional support of families of advanced cancer patients: A nationwide survey of bereaved family members

Background: A number of advanced cancer patients are suffering from physical and psychosocial burdens because of cancer cachexia, and these burdens also greatly impact on their family members and relationships between patients and family members. It is necessary to consider the psychosocial impact of cancer cachexia on family members of advanced cancer patients. Methods: A cross-sectional anonymous nationwide survey was conducted involving 925 bereaved family members of cancer patients who had been admitted to 133 inpatient hospices throughout Japan. Results: A total of 702 bereaved family members returned the questionnaires (response rate, 75.9%). Concerning eating-related distress, 'I served what the patient wanted without consideration of calories and nutritional composition' was highest (75.1%), and 'I tried making many kinds of meals for the patient' and 'I was concerned about planning meals for the patient every day' followed (63.0% and 59.4%, respectively). The top 5 of the 19 items were categorized as 'fighting back'. Need for nutritional support was high (72.2%), and need for explanations about the reasons for anorexia and weight loss of patients was moderate (41.4%). Explanatory factor analysis of eating-related distress identified the following four domains: (factor 1) feeling that family members forced the patient to eat to avoid death, (factor 2) feeling that family members made great efforts to help the patient eat, (factor 3) feeling that eating was a cause of conflicts between the patient and family members, and (factor 4) feeling that correct information was insufficient. Results of multiple logistic regression analysis showed that spouse, fair/poor mental status, factors 1, and 4 were identified as independent determinants of major depression (odds ratio [OR] 3.27 [95% confidence interval (CI) 1.24-8.60], P=0.02; OR 4.50 [95% CI 2.46-8.25], P<0.001; OR 2.51 [95% CI 1.16-5.45], P=0.02; OR 2.33 [95% CI 1.13-4.80], P=0.02, respectively). Conclusions: A number of family members of advanced cancer patients experienced high levels of eating-related distress and had a need for nutritional support

A study on the time-matching in three dimensional cinematography

本研究の目的は,同期しているデータと同期上の誤差を含んだデータの解析結果を比較し,民生用ビデオの定量的動作解析への利用の可能性を検討することであった。フレームカウンターを接続した2台のビデオカメラにより,バレーボールのスパイク動作を撮影した。2台のカメラの2次元座標が同期している場合,No.1カメラとNo.2カメラの2次元座標の時刻が1/120秒ずれている場合,No.1カメラとNo.2カメラの2次元座標の時刻が1/60秒ずれている場合の3通りの同期誤差をもつ2次元座標を準備し,DLT法により身体各部位及びボールの3次元座標を算出した。スティックピクチャーによる視覚的比較,各種測定項目による数値的比較を通して,同期,同期誤差1/2field,同期誤差1fieldのデータ間に大差が見られなかったこと,そして大部分の測定項目の値は先行研究の値の範囲にあったことから,民生用ビデオによる3次元動作解析は可能であると考えられた。The purpose of this study was to compare the analysis results of Time-Matching with Time-Error data and to find the possible utilization for quantitative motion analysis by home videos.It took spiking motions of volleyball by two video cameras with frame counter. After prepared three cases of two-dimensional coordinates that the time of two cameras are matching, different of 1/120 and 1/60 seconds, three-dimensional coordinates of the segment endpoints and a ball were calculated by Direct Linear Transformation Method.There were no great difference in Time-Matching, 1/2 and 1field data through the visual comparison by stick-pictures and the numerically comparison by various kinds of items of measurement. Consequently, the results suggest that it is possible to do three dimensional motion analysis by home videos

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules