Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Improvement of Storage and Deastringency Technology and Studies on the Deastringency Mechanism of Persimmon (Diospyros kaki L.) Fruits

柿子葉可溶性單寧濃度因品種而異，本調查中以‘牛心柿’最高，其次為‘花御所’，再者‘富有’、‘蜂屋’及‘平核無’，三者間差異不大；葉片中PAL活性和可溶性單寧濃度變化趨勢一致。柿葉經二氧化碳處理後可溶性單寧呈下降趨勢，和果實一樣有脫澀情形，似可作為研究脫澀之材料。柿果發育中生長曲線呈雙S型曲線，幼果期‘牛心柿’即有較高的可溶性單寧濃度，且成長中單果含量有累積現象，而‘富有’柿可溶性單寧含量及濃度皆低，且其PAL活性亦較‘牛心柿’低，由結果顯示，‘富有’柿合成單寧能低，加上果實成長之稀釋效應，至採收時已不具澀味。 ‘牛心柿’以各種脫澀方法處理時，其脫澀速度由快而慢依序為二化碳脫澀法、酒精脫澀法、石灰懸浮液脫澀法及益收脫澀法。100%氮氣處理時柿果果肉亦可和二氧化碳處理一樣累積酒精和乙醛，但脫澀效果不及二氧化碳處理者，而酒精處理時，以電泳分析酒精去氫酵素(ADH)之表現，在處理後60小時才見ADH條帶出現，但柿果於12小時後即開始脫澀，結果顯示，乙醛可能非促成脫澀之主要因子。在果肉圓片系統下，二氧化碳處理時，Cycloheximide及Tween-60不會抑制脫澀，Aminooxyacetic acid，Sodium cyanide及熱處理會抑制脫澀；另外，完整果實在脫澀時皆可見果實硬度下降，由此結果推測，柿果脫澀處理時，有一非附膜且需pyridoxal phasphate酵素被活化，而催化細胞壁水解產生滲透性脫水，促使脫澀。 ‘牛心柿’在6℃低溫貯藏時，果皮由黃綠色轉橙黃色，果肉較暗呈水浸狀，硬度下降及不易脫澀等寒害症狀。本試驗主要探討‘牛心柿’果實於低溫貯藏前，利用溫湯處理，對果實品質之影響。‘牛心柿’先以溫湯處理，再低溫貯藏(6℃) 30天後，各處理以酒精脫澀處理3天，有脫澀不完全的情形；貯藏60天後澀味指數經3天酒精脫澀處理，澀味指數可降至1，可能和果肉軟化有所關聯，且有寒害症狀產生；其中以48℃熱水處理30分鐘及50分鐘可維持較高的果肉硬度及品質。 本試驗將‘筆柿’以不同脫澀方法處理，以了解‘筆柿’脫澀所需之最適條件及觀察物理性狀和化學組成分之變化。‘筆柿’以10ppm乙烯催熟，在20-30℃下後熟，3日可完全脫澀，果實顏色由黃橙變紅呈現橙紅色；酒精脫澀處理，在30℃下，每公斤柿果需6ml之酒精量，於3日後可完全脫澀。果實以酒精及二氧化碳處理，脫澀之速度隨溫度增加而加速，25℃下需5日以上，30及35℃需4-5日，而在40℃下則只需2-3日即可完成脫澀，但以30及35℃脫澀後之品質較佳。The concentrations of the soluble tannin in persimmon leaves differed markedly in different varieties. ‘Bull Heart' and ‘Hanagoshiyo' leaves had particularly high concentrations of soluble tannin while no significant differences were found among ‘Fuyu', ‘Hachiya' and ‘Hiratanenashi'. Similar trends were observed for PAL activity as in the case of soluble tannin. Like fruit deastringency, the concentration of soluble tannin in leaves decreased after carbon dioxide treatment. Thus the leaves could be used as material to investigate the mechanism of deastringency. The curve of fruit development in ‘Fuyu' and ‘Bull Heart' persimmon tended to be a double sigmoid. At the young fruit stage, the PAL activity and tannin content of ‘Bull Heart' fruits were higher than those of ‘Fuyu' fruits and had the tendency of accumulation during growth and development. Meanwhile, soluble tannin content of the ‘Fuyu' fruit was low and the PAL activity was also lower than the ‘Bull Heart'. The results indicated that tannin synthesis ability of ‘Fuyu' and the dilution effects of fruit growth were responsible for the low astringency of fruits at harvest. In vivo and whole fruit studies were conducted on the effectiveness of deastringency of persimmon “Bull Heart” fruit. Carbon dioxide treatment was found to have higher deastringency rate than alcohol, CaO suspension and ethrel. Further studies showed that fruits treated with 100% nitrogen, as in the case of carbon dioxide, accumulated acetaldehyde and ethanol in fruits but the rate of deastringency was low and the method was less effective. While removal astringency with alcohol, electrophoresis bands of alcohol dehydrogenase (ADH) appeared at 60 hours during deastringency period, but the deastringency reactions of the fruit were observed 12 hours after the treatment, which indicates that acetaldehyde is not a key factor in the process. Fruit discs were also used in the deastringency experiments of ‘Bull Heart'. Soaking discs in various solutions and then treating with carbon dioxide and revealed that deastringency was inhibited by aminooxyacetic acid, sodium cyanide and hot water treatments (70-100℃for 10 min) but not by cycloheximide and Tween-60. This indicates that deastringency of persimmon requires the activation of a non-peripheral and pyridoxal-phosphate dependent enzyme, which catalyzes cell wall hydrolysis and leads to the osmosis dehydration which is followed by triggering the polymerization of tannin to complete the process. Persimmon fruits ‘Bull Heart' when stored at 6℃, the peel color turned from yellow-green to orange-yellow, and the color of pulp became dark and water soaked. Firmness decreased and astringency could not be eliminated. Those symptoms were confirmed to be chilling injuries. The objectives of this study were to evaluate the effects of warm-water treatment before cold storage (6℃) on the qualities of the fruits. ‘Bull Heart' treated with warm water and stored in cold storage for 30 days, followed by 3-day alcohol treatment resulted in an incomplete deastringency; The astringent index was down to 1 after 60 days of cold storage and 3 days of alcohol treatment. This could relate to the softening of the fruits and chilling injury. Warm-water treatments (48℃-30min and 48℃-50min) reduced the damage of chilling injury and retained the firmness and quality of fruits. The effects of different deastringent treatments on physical character and chemical compositions changes of the ‘Bi-Su' persimmon were evaluated in this experiment. The astringency of persimmon fruits was completely removed by 10 ppm ethylene together with after-ripening for 3 days at 20-30℃. The peel color turned from yellow-green to red after the completion of deastringency. Ethanol treatment at the dosage of 6ml/kg completely eliminated the astringency after 3 days at 30℃. The effects of temperature on ‘Bi-Su' persimmon fruits treated with carbon dioxide and ethanol were examined during the process of deastringency at various temperature ranging from 20-40℃. The time required to reach astringent index 1 were 2-3, 4-5 and more than 5 days at 40℃, 30-35℃and 20-25℃, respectively. The deastringency temperatures required for the better fruits qualities were 30-35℃.目錄 壹、 前人研究 1 一、 柿之概說 1 二、 果實之生長發育 2 三、 苯丙胺酸解氨酶在果實生長發育及採後之變化 3 四、 柿果的脫澀方法 5 五、 柿果脫澀機制 7 六、 低溫貯藏前熱處理對果實寒害之影響 10 七、 乙烯與果實後熟軟化 12 貳、 柿子葉片之脫澀處理及柿果生長發育期間苯丙胺酸解氨酶與可溶性單寧含量之變化 15 摘要 15 SUMMARY 16 一、 前言 17 二、 材料與方法 18 三、 結果與討論 20 參、 ‘牛心柿’不同脫澀處理脫澀之機制 29 摘要 29 SUMMARY 30 一、 前言 31 二、 材料與方法 32 三、 結果 37 四、 討論 53 肆、 低溫貯藏前溫湯處理對‘牛心柿’脫澀品質之影響 59 摘要 59 SUMMARY 60 一、 前言 61 二、 材料與方法 62 三、 結果 63 四、 討論 76 伍、 ‘筆柿’脫澀處理條件之研究 79 摘要 79 SUMMARY 80 一、 前言 81 二、 材料與方法 82 三、 結果 84 四、 討論 112 參考文獻 11

Effect of Carbon Dioxide Treatment on the Deastringency of 'Bull Heart' Persimmon Fruits

本試驗之目的在調查，牛心柿．經過不同二氧化碳濃度處理後，對果實品質之影響及生理之變化。結果顯示，'牛心柿'果實經氣體脫澀後，各處理組澀味指數皆下降，其中以二氧化碳處理者處理3天後，下降最多;對照於可溶性單寧含量以二氧化碳處理者皆低於0.5%，且柿果所含可溶性固形物降低，主要可能來自於可溶性單寧含量下降引起。脫澀後各處理組較對照組可測得較高含量的酒精及乙醛，其中100%氮氣處理時柿果果肉亦可和二氧化碳處理組一樣累積酒精和乙醛，甚至高於25%二氧化碳處理者，但脫澀效果卻不及二氧化碳處理者，結果顯示，乙醛可能非促成脫澀之主要因子。The objective of this studied were to investigate the effect of carbon dioxide and nitrogen treatment on the quality and physiology change of ‘Bull Heart'persimmon fruits. The results indicated the persimmon fruits deastringency with carbon dioxide and nitrogen which tended to have a reduce of astringency index and soluble tannin concentration, and the carbon dioxide deastringency treatment fruits which may resulted from the reduced of soluble tannin. Further studied showed that fruits treated with 100％ nitrogen, as in the case of carbon dioxide, accumulated acetaldehyde and ethanol in fruits but the rate of deastringency was low and the method was less effective. Results indicated that acetaldehyde is not a key factor in the process

Up-Regulation of CYP2C19 Expression by BuChang NaoXinTong via PXR Activation in HepG2 Cells.

Cytochrome P450 2C19 (CYP2C19) is an important drug-metabolizing enzyme (DME), which is responsible for the biotransformation of several kinds of drugs such as proton pump inhibitors, platelet aggregation inhibitors and antidepressants. Previous studies showed that Buchang NaoXinTong capsules (NXT) increased the CYP2C19 metabolic activity in vitro and enhanced the antiplatelet effect of clopidogrel in vivo. However, the underlying molecular mechanism remained unclear. In the present study, we examined whether Pregnane X receptor (PXR) plays a role in NXT-mediated regulation of CYP2C19 expression.We applied luciferase assays, real-time quantitative PCR (qPCR), Western blotting and cell-based analysis of metabolic activity experiments to investigate the NXT regulatory effects on the CYP2C19 promoter activity, the mRNA/ protein expression and the metabolic activity.Our results demonstrated that NXT significantly increased the CYP2C19 promoter activity when co-transfected with PXR in HepG2 cells. Mutations in PXR responsive element abolished the NXT inductive effects on the CYP2C19 promoter transcription. Additionally, NXT incubation (150 and 250μg/mL) also markedly up-regulated endogenous CYP2C19 mRNA and protein levels in PXR-transfected HepG2 cells. Correspondingly, NXT leaded to a significant enhancement of the CYP2C19 catalytic activity in PXR-transfected HepG2 cells.In summary, this is the first study to suggest that NXT could induce CYP2C19 expression via PXR activation

NXT induced the CYP2C19 protein expression in HepG2-PXR cells.

<p>HepG2 cells were transiently transfected with pcDNA3.1-PXR for 24 hours and then incubated with 0.1% DMSO, 10μM rifampicin, 150 and 250μg/mL NXT for 24 hours, respectively. (A) the transfection efficiency of pcDNA3.1-PXR in HepG2 cells; (B) expression of CYP2C19 protein analyzed by Western blotting; (C) accurate induction of NXT on CYP2C19 protein were quantified by densitometry. Rif: rifampicin; NXT-150 and -250: 150 and 250μg/mL NXT; Data was presented as the mean±S.D from three independent experiments. *P<0.05,**P<0.01 vs. DMSO group.</p