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 flux 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, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

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

With digital equipment becoming increasingly networked, either on wired or wireless networks, for personal and professional use alike, distributed software systems have become a crucial element in information and communications technologies. The study of these systems forms the core of the ARLES' work, which is specifically concerned with defining new system software architectures, based on the use of emerging networking technologies. In this context, we concentrate on the study of distributed systems which bring to life the vision of ubiquitous computing systems, also known as ambient intelligence.

반도체 공정 overview

正しい方法がわかる臨床基本手技 : from the New England journal of medicine

Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

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AMPK: guardian of metabolism and mitochondrial homeostasis.

Autophagy and apoptosis control the turnover of organelles and proteins within cells, and of cells within organisms, respectively, and many stress pathways sequentially elicit autophagy, and apoptosis within the same cell. Generally autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, in special cases, autophagy or autophagy-relevant proteins may help to induce apoptosis or necrosis, and autophagy has been shown to degrade the cytoplasm excessively, leading to 'autophagic cell death'. The dialogue between autophagy and cell death pathways influences the normal clearance of dying cells, as well as immune recognition of dead cell antigens. Therefore, the disruption of the relationship between autophagy and apoptosis has important pathophysiological consequences.

Self-consumption: the interplay of autophagy and apoptosis

Hypothesis / aims of study: Cyclophosphamide (CPO) and ifosfamide (IFO) are commonly used anticancer and immunosuppressive agents. A major limiting factor in the use of CPO and IFO is the resulting uro-toxicity thought to be caused, in part, by reactive oxygen species formation and resulting in ongoing bladder pain, urgency and dysuria. The uro-toxicity of these drugs has been largely attributed to the formation of the metabolite acrolein. However another toxic metabolite chloroacetaldehyde, has been implicated in the neuro-, cardioand nephro-toxicity of these drugs, but possible uro-toxicity has not been well investigated. These drugs and their metabolites are excreted in the urine and come into contact with the urothelium. The urinary concentration of acrolein or chloroacetaldehyde likely to occur in patients is 100nM or 10uM respectively (1-3). It is well known that the urothelium plays an important role in maintaining normal bladder function, releasing a number of mediators (eg. ATP and acetylcholine) that can influence sensory nerve sensitivity and detrusor muscle contraction. Accordingly, this study aimed to investigate the affects of the CPO and IFO metabolites acrolein and chloroacetaldehyde on human urothelial cell viability and function in vitro.

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Chloroacetaldehyde, and not acrolein, is the more uro-toxic metabolite of cyclophosphamide and isofosfamide in vitro

Interstitial cystitis/bladder pain syndrome (IC/BPS) remains a clinical enigma. Estimated to affect more than 8 million women globally (3, 19), it is a challenge to diagnose and to treat effectively due to an unknown etiology and complex, poorly understood pathophysiology. Clinical diagnosis is symptom based, with patients experiencing debilitating chronic pelvic or bladder pain, pressure, or discomfort, along with urinary storage symptoms, including frequency and urgency (10). IC/BPS is also associated with depression and anxiety (8). Current treatments for IC/BPS are varied and include diet and lifestyle changes, intravesical instillations directly into the bladder, oral medications, and, as a last resort, surgery. Generally, success rates for therapeutic interventions are modest at best, and there is little consensus with respect to which is the most effective. Intravesical dimethyl sulfoxide therapy has been widely used for decades, alone or as part of a cocktail in combination with other agents such as local anesthetics (15). However, long-term outcomes are poorly documented. In a recent study, intravesical DMSO/heparin/hydrocortisone therapy was only moderately effective for treatment of IC/BPS, with only half (56%) of patients well or significantly improved at 5 yr (13). A better understanding of the pathophysiology of this condition could potentially lead to the identification of novel pharmacological targets and the development of more successful treatments. As such, there has been extensive research focused on unraveling the underlying mechanisms of IC/BPS. Locally within the bladder wall, known key pathological changes include urothelium disruption, impaired barrier function, and inflammation. These, in addition to sensitized peripheral and central neural pathways, all contribute to pain and urinary symptoms (15). There is also convincing epidemiological evidence supporting the link between IC/BPS and a history of urinary tract infection (16) and activation of the innate immune system. Toll-like receptors (TLR) are a family of membrane glycoproteins expressed primarily on cells of the immune system that act as pattern recognition molecules. They can bind pathogen-derived molecules and mediate signaling cascades, leading to an inflammatory response (12). Our understanding of TLRs has expanded greatly from their role in immune cells into their role in nonimmune cells and tissues, and they have become an area of recent interest in inflammatory disorders of the lower urinary tract. Although there has been some focus on TLR7 in IC/BPS, where TLR7 expression was found to be upregulated in bladder biopsies from Hunner-type (ulcerative) IC patients, and a TLR7 agonist increased expression of TLR7 mRNA and induced cystitis in mice (11), the field is not well researched. TLR4 is expressed on urothelial cells (21), and its role in mediating inflammation following infection of the bladder has been well established for some time (2). Schrepf et al. (22) have shown that TLR4 inflammatory responses in peripheral blood mononuclear cells are a marker of widespread pain in IC/BPS. However, until now the role of TLR4 in IC/BPS was not well understood. In a recent issue of the American Journal of PhysiologyRenal Physiology, de Oliveira et al. (9) elegantly demonstrate for the first time, and at both a molecular and functional level, that TLR4 plays a central role in initiating cyclophosphamideinduced bladder dysfunction and inflammation. In this study, convincing evidence is provided that genetic deletion using TLR4 / mice or pharmacological inhibition of TLR4 with the selective antagonist resatorvid prevents dysfunction in micturition and inflammatory events associated with cystitis. These data directly demonstrate for the first time important etiological information regarding CYP-induced cystitis in the increased expression of TLR4 downstream signaling MyD88 and TRIF, which supports an important role for this receptor. A key finding that is possibly somewhat underplayed is the identification of a role for TLR4 in normal bladder function shown in the untreated TLR4 / mice, which displayed different voiding profiles with increased frequency of nonvoiding contractions. This suggests that TLR4 signaling impacts the afferent arm of micturition, and it will be interesting in future studies to determine whether this is via a direct effect on afferent nerves per se or an indirect effect via the release of mediators from the urothelium, which is a source of inflammatory peptides and cytokines (14). Because other endogenous ligands can activate TLR4, including ECM components, heat shock proteins, and -defensins (18), other physiological roles for TLR4 in normal bladder control may yet be uncovered. A confounding factor in our progress in understanding the pathophysiology of IC/BPS has been the limitations of the different animal models employed, which was recently brought to the forefront in an excellent review by Birder and Andersson (4). IC/BPS is complex and there are as yet no animal models available that mimic all aspects of the human experience. In the study by de Oliveira et al.(9), the well-established cyclophosphamide (CYP)-induced mouse model of acute cystitis was used, in which urotoxicity results in pain-related behavior, altered micturition patterns, and bladder overactivity as well as inflammation (5, 6), all traditional features of ICS/BPS. Although it is important to acknowledge the limitations of such animal models, they remain important for the elucidaAddress for reprint requests and other correspondence: D. J. Sellers, Centre for Urology Research, Faculty of Health Sciences and Medicine, Bond University, University Dr., Robina, Queensland 4229, Australia (e-mail: dsellers @bond.edu.au). Am J Physiol Renal Physiol 315: F910–F912, 2018; First published June 27, 2018; doi:10.1152/ajprenal.00290.2018.

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A central role for Toll-like 4 receptors in interstitial cystitis?

Evidence to support the effectiveness of β3-adrenoceptor agonist mirabegron and anti-muscarinic solifenacin in the management of bladder dysfunction caused by psychological stress is lacking. This study investigates whether mirabegron or solifenacin reduces the bladder overactivity caused by water avoidance stress (WAS) in mice. Female mice were exposed to WAS for 1 h/day for 10 days and received either placebo, solifenacin or mirabegron in drinking water. Controls were age-matched without stress exposure. Voiding behaviour and functional isolated whole bladder responses during distension and in response to pharmacological agents and electrical field stimulation was investigated. Urinary frequency was significantly increased following stress. Mice treated with mirabegron or solifenacin displayed significantly fewer voiding events compared to the stressed mice, and voiding frequency in drug-treated animals was comparable to unstressed controls. The maximal contractile responses of bladders to carbachol were significantly enhanced by stress and reduced by mirabegron but not solifenacin. The frequency of phasic bladder contractions following stimulation with carbachol was significantly enhanced following stress and remained elevated in the mirabegron treated group. However, treatment with solifenacin significantly reduced the frequency of phasic contractions to unstressed control levels. Solifenacin and mirabegron are beneficial in reducing the overall voiding dysfunction caused by WAS in mice. 

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Catherine McDermott

Papers

Chloroacetaldehyde, and not acrolein, is the more uro-toxic metabolite of cyclophosphamide and isofosfamide in vitro

A central role for Toll-like 4 receptors in interstitial cystitis?

Mirabegron and solifenacin are effective for the management of the increased urinary frequency induced by psychological stress in female mice